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Chapter 1
An Introduction to Enzyme Science
Enzymes are astonishing catalysts – often achieving rate
enhancement factors1 of 1,000,000,000,000,000,000!
Water, electrolytes, physiologic pH, ambient pressure and
temperature all conspire to suppress chemical reactivity to
such a great extent that even many metabolites as thermodynamically unstable as ATP (DGhydrolysis z 40 kJ/mol)
and acetyl-phosphate (DGhydrolysis z 60 kJ/mol) are inert
under normal physiologic conditions. Put simply, metabolism would be impossibly slow without enzymes, and Life,
as we know it, would be unsustainable.2 As a consequence,
enzymes are virtual on/off- switches, with efficient
conversion to products in an enzyme’s presence and
extremely low or no substrate reactivity in an enzyme’s
absence. At millimolar concentrations of glucose and
MgATP2, for example, substantial phosphorylation of
glucose would require hundreds to thousands of years in the
absence of hexokinase, but only seconds at cellular
concentrations of this phosphoryl transfer enzyme. Without
hexokinase, there would also be no way to assure exclusive
phosphorylation at the C-6 hydroxymethyl group. And even
when an uncatalyzed reaction (termed the reference
1
Catalytic rate enhancement (symbolized here as 3) equals the unit-less
ratio kcat/kref, where the catalytic rate constant kcat (units ¼ s1) is the
catalytic frequency (i.e., the number of catalytic cycles per second per
enzyme active site), and kref (units ¼ s1) is the corresponding
first-order rate constant for the uncatalyzed reaction. The value of 3 will
be a direct measure of catalytic proficiency (i.e., an enzyme’s ability to
enhance substrate reactivity), if and only if the enzymatic and
nonenzymatic reactions operate by the very same chemical mechanism,
in which case the nonenzymatic reaction is called the reference
reaction. Note also that the value of 3 achieved by any given enzyme
need only be sufficient to assure unimpeded metabolism. In the
Principle of Natural Selection, mutation is the underlying search
algorithm for evolution, and any mutation that markedly improves 3
beyond that needed for an organism’s survival should be inherently
unstable and subject to reduction over time.
2
The upper limit on the room temperature rate constant for nonenzymatic
water attack on a phosphodiester anion, for example, is about 1015 s1,
necessitating 100-million year period for uncatalyzed P–O cleavage
(Schroeder et al., 2006). Depending on reaction conditions, the
corresponding rate constant for hydrolysis of the bg P–O bond in
MgATP2 is around 104 to 106 s1, and given that bimolecular
processes obey the simple rate law v ¼ k[A][B], rates for phosphoryl
group transfer reactions (e.g., MgATP2 þ Acceptor # Phosphoryl Acceptor þ MgADP) would be suppressed even further at low
micromolar-to-millimolar concentrations of acceptor substrates within
most cells.
Enzyme Kinetics
Copyright Ó 2010, by Elsevier Inc. All rights of reproduction in any form reserved.
reaction) is reasonably fast – as is the case for the reversible
hydration of carbon dioxide to form bicarbonate anion or for
the spontaneous hydrolysis of many lactones – an enzyme
(in this case, carbonic anhydrase) is required to assure that
the reaction’s pace is compatible with efficient metabolism
under the full range of conditions experienced by that
enzyme. Most enzymes also exhibit rate-saturation kinetics,
meaning that velocity ramps linearly when the substrate
concentration is below the Michaelis constant, and reaches
maximal activity when the substrate is present at a concentration that is 10–20 times the value of the Michaelis
constant. In this respect, an enzyme’s action is more akin to
a variable-voltage rheostat than a simple on/off switch.
Biochemists recognize that substrate specificity is
another fundamental biotic strategy for effectively organizing biochemical reactions into metabolic pathways. Two
analogous chemical reactions can take place within the
same (or adjoining) subcellular compartments simply
because their respective enzymes show substrate or cofactor
specificity directing metabolic intermediates to and through
their respective pathways, often without any need for subcellular co-localization or enzyme-to-enzyme channeling.
Substrate specificity also minimizes formation of unwanted,
and potentially harmful, by-products. By controlling the
relative concentrations of such enzymes, cells also avoid
undesirable kinetic bottlenecks or the undue accumulation
of pathway intermediates.3 Experience tells us that
extremely reactive chemical species can also be sequestered
within the active sites of those enzymes requiring their
3
The term intermediate has several distinctly different meanings in
biochemistry. In the context of the above sentence, intermediate refers
to a chemical substance that is produced by an enzyme reaction within
a metabolic pathway (A / B / C / P / Q / R, where B, C, P,
and Q are metabolic intermediates) and is likewise a substrate in
a subsequent enzyme-catalyzed reaction in that or another pathway. In
the very next sentence, intermediate refers to a enzyme-bound substrate,
enzyme-bound reactive species, or enzyme-bound product formed
during the catalysis (E þ S # ES1 # ES2 # EXz # EP1 # EP2 #
E þ P, where ES1, ES2, EXz, EP1, and EP2 are various enzyme-bound
species/intermediates) in a single enzymatic reaction. For reactions
occurring in the absence of a catalyst, chemists routinely use the term
intermediate to describe any reactive species Xi-1, formed during the
course of chemical transformation, whether formed reversibly (i.e.,
Xi-1 # Xi # Xiþ1) or irreversibly (i.e., Xi-1 / Xi / Xiþ1). All such
usages of intermediacy connote metastability and/or a transient nature.
1
Enzyme Kinetics
2
formation, while hindering undesirable side-reactions that
would otherwise prove to be toxic. So enzyme catalysis is
inherently tidy. Enzyme active sites can also harbor metal
ions that attain unusually reactive oxidation states that rarely
form in aqueous medium and even less often in the absence
of side-reactions. The resilience of living organisms stems in
large measure from the capacity of enzymes to specifically or
selectively bind other ligands (e.g., coenzymes, cofactors,
activators, inhibitors, protons and metal ions).
Attesting to the significance of enzyme stereospecificity in
the biotic world is that most metabolites and natural products
contain one or more asymmetric carbon atoms. The stereospecific action of enzymes is the consequence of the fact that
both protein and nucleic acid enzymes are polymers of
asymmetric units, making resultant enzymes intrinsically
asymmetric. It should be obvious that any L-amino acidcontaining polypeptide having even a single D-amino acid
residue cannot adopt the same three-dimensional structure as
a natural polypeptide. Although some enzymes utilize both
enantiomers of a substrate (e.g., glutamine synthetase is
almost equally active on D-glutamate and L-glutamate),
proteins containing exclusively L-amino acids are produced
by the ribosome’s peptide-synthesizing machinery. This
outcome is the result of the stereospecificity of aminoacyltRNA synthases that supply ribosomes with activated
subunits, the stereochemical requirements of peptide
synthesis, as well as ubiquitinylating enzymes and proteasomes that respectively recognize and hydrolyze wrongly
folded proteins. Cells also produce a range of enzymes, such
as D-amino acid oxidase (Reaction: D-Amino Acid þ O2 þ
H2O # 2-Oxo Acid þ NH3 þ H2O2), that remove certain
enantiomers (in this case, D-amino acids) from cells. In the
case of protein enzymes, certain aspartate residues are also
susceptible to spontaneous racemization as well as N-to-O
acyl shifts, and cells produce enzymes that recognize and
mediate the repair or destruction of proteins containing
monomers having improper stereochemistry.
Additional metabolic pathway stability is afforded by
steady-state fluxes that resist sudden changes in rate or
reactant concentrations. The processes lead to the phenomenon of homeostasis, wherein reactant concentrations appear
to be time invariant merely because the processes producing
and destroying these reactants are so exquisitely controlled.
In some respects, the behavior of the whole of metabolism
appears to exceed the sum of behaviors of its individual
reactions. Experience has shown that hierarchically complex,
large-scale networks often give rise to emergent properties
(i.e., properties of a highly integrated metabolic or physiologic system that are not easily predicted from the analysis of
individual components). Beyond the coordinated operation
and regulation of the many pathways comprising intermediary metabolism, other emergent properties of living
systems are evident in the adaptive resilience of signal
transduction, long-range actions affecting chromosomal
organization, as well as cellular morphogenesis and motility.
The creation of organizationally complex neural networks, as
facilitated by the capacity of single neuronal cells to engage
in tens of thousands of cell–cell interactions with other
neurons via synapse formation, is also thought to underlie
what we sense as our own consciousness. And at all such
levels, enzyme catalysis and control are inevitably needed for
effective intracellular and intercellular communication.
As the essential actuators of metabolism, enzymes are
often altered conformationally via biospecific binding
interactions with substrates and/or regulatory molecules
(known as modulators or effectors) to achieve optimal
metabolic control. An additional feature is the capacity of
multi-subunit enzymes to exhibit cooperativity (i.e.,
enhanced or suppressed ligand binding as a consequence of
inter-subunit cross-talk). Because enzyme structure changes
can be triggered by changes in the concentrations of
numerous ligands, enzymes possess an innate capacity to
integrate diverse input signals, thereby generating the most
appropriate changes in catalytic activity. An interaction is
said to be allosteric if binding of a low-molecular weight
substance results in a metabolically significant conformational change. In most cases, modulating effects are negative (i.e., they result in inhibition), but positive effects (i.e.,
those resulting in activation) are also known. Feedback
regulation has proven to be a highly effective strategy for
controlling the rates of metabolic processes. When present
at sufficient concentration, a downstream pathway intermediate or product (known as a feedback inhibitor) alters
the structure of its target enzyme to the extent that the
inhibited enzyme exhibits little ot no activity (Scheme 1.1).
Target enzymes (shown below in red) are most often positioned at the first committed step within a pathway or at
a branch point (or node) connecting two or more pathways.
The lead reactions are frequently highly favorable (DG <<
0), whereas the intervening reactions are generally reversible (DG ¼ 0), or nearly so (DG z 0).
EE F F
A
EA
B
C
EB
D
EC
EG
E
ED
EE I
EJ
EI
I
H
G
EF
J
K
Scheme 1.1
Feedback inhibition (shown in blue) of target enzymes
therefore precludes unnecessary accumulation of possibly
toxic metabolic pathway intermediates. By contrast, elevated
metabolic throughput (or flux) is observed when an enzyme
responds to an allosteric activator. In the latter case, the
enzyme achieves no or partial catalytic activity in the absence
of an activator, and biospecific binding of the activator alters
the target enzyme’s conformation in a way that increases its
catalytic efficiency. Although the hallmark of allosteric
Chapter j 1 An Introduction to Enzyme Science
enzymes is cooperativity (i.e., subunit–subunit interactions
altering the apparent substrate binding affinity), metabolic
control is also achieved by the regulated synthesis and
degradation of specific enzymes, by interconversion between
enzyme activity states via enzyme-catalyzed covalent modification, by effector molecule mediated signal amplification,
and in some instances by substrate channeling.
Molecular life scientists have uncovered countless
instances wherein improper catalysis and/or regulation of
even a single enzyme reaction can greatly distress a living
organism. Such mutant enzymes wreak havoc on cellular
physiology. In fact, animal and plant diseases frequently
arise from point mutations that result in site-specific
substitution of a single amino acid residue in an enzyme.
Elaborate proofreading mechanisms permit replication,
transcription, and translation to proceed at rapid rates, while
minimizing error propagation, and a battery of repair
enzymes correct DNA damage arising unavoidably from
photolysis, oxidation, alkylation, hydrolysis, and racemization. The same is true of errors occurring during the
synthesis, splicing, and turnover of RNA transcripts. Ribosomes must also occasionally commit errors, but with the
possible exception of prion protein formation the impact of
low-level occurrence of ‘‘translational mutations’’ is apt to
be minimal. Other more injurious mistakes made during
replication and transcription are known to culminate in
enzyme over-/under-production, defective regulation,
impaired stability, incorrect post-translational modification,
improper subcellular targeting and compartmentalization,
defective turnover, etc. A notable example is amyotrophic
lateral sclerosis or ALS (widely known as Lou Gehrig’s
disease). This devastating neurodegenerative disorder is
linked to the impaired action of superoxide dismutase; overaccumulation of superoxide (O2) damages neurons, an
injury that is attended by profound pathological sequelae.
Another example is the discovery that Pin1-catalyzed cistrans prolyl residues isomerization can alter the structure of
the microtubule-associated protein Tau in axons and that
Pin1 gene knockouts bring about progressive age dependent
neuropathy characterized by motor and behavioral deficits,
attended by hyper-phosphorylation of Tau, as well as Tau
polymerization into neurodegenerative paired helical filaments (Liou et al., 2003). Although more research is
required to assess the significance of such findings to the
onset of Alzheimer’s disease, it is already clear that reduced
prolyl cis-trans isomerization activity can profoundly
impair neuronal function.
Enzyme chemists investigate biological catalysis by
assessing the structural and energetic features of the
elementary reactions comprising a multi-step enzyme
mechanism. They seek to understand how activators and
inhibitors alter the energetics of catalytic reaction cycles to
bring about effective metabolic regulation. The daunting
task of determining how an enzyme operates is never an
easy matter, and without a systematic approach, one is
3
forced to glean information haphazardly. A more effective
strategy starts with a reliable assay of catalytic activity and
requires the experimenter to use this assay in the isolation of
the enzyme of interest from other contaminants (e.g.,
proteins, solutes, etc.) affecting the enzyme’s activity. In
practice, absolute purity is not required as long as other
contaminating enzymes and proteins are without effect on
the enzyme of interest. It is helpful to apply the principles of
organic chemistry to infer likely chemical transformations
occurring during catalysis, using literature precedents to
guide one’s thoughts about the roles of coenzymes and
cofactors and to focus on probable reaction intermediates.
Ultimately, however, it is necessary to test whether each
reaction step occurs on a time-scale consistent with its role
in catalysis. This latter pursuit, called enzyme kinetics,
combines an interest analyzing temporal aspects of enzyme
catalysis with the principles of physical chemistry and
quantitative rigor of analytical chemistry.
Some of the stages in the characterization of a complete
enzyme mechanism are listed in Fig. 1.1. Because initial-rate
kinetics is a relatively straightforward tool for analyzing
enzyme catalysis, we may regard such experimental
approaches as the first stage in the systematic characterization of an enzyme of interest. Pursuit of subsequent stages
depends on the objectives of the particular investigation.
This reference explains how enzyme kineticists formulate
and test models to: (a) explain the reactivity and energetics
of enzyme processes; (b) gain the most complete description
Stage-1: Initial Rate Kinetics
v versus [substrate(s)] → Km,Vm & VmIKm
Substrate Specificity & Side-Reactions
Product Inhibition → Substrate Binding Order
Competitive Inhibition → Substrate Binding Order
pH Kinetics → pK’s of Catalytic Groups
Site-Directed Mutagenesis
Stage-2: Chemical Studies
Determination of Reaction Stereochemistry
Detection of Tightly Bound Coenzymes & Metal Ions
Detection of Covalent Intermediates
Identification of Active-Site Residues by Affinity Labeling
Stage-3: Isotope Kinetics
Partial Exchange Reaction → Substrate Binding Order
Isotope Exchange at Equilibrium → Substrate Binding Order
Isotope Trapping & Partition Kinetics → “Stickiness”
Positional Isotope Exchange → Reaction Intermediates
Kinetic Isotope Effects → Reaction Intermediates
Stage-4: Fast Reaction Kinetics
Continuous, Stopped-Flow & Mix/Quench Techniques
Temperature-Jump & Pressure-Jump Techniques
Stage-5: Single-Molecule Reactions
Reaction Trajectories
Mechanochemistry of Force Generation
FIGURE 1.1 Kinetic tools in modern enzyme science. Depicted here
are the typical stages in order of complexity for the characterization of
an enzyme-catalyzed reaction. Within each stage are various experimental
approaches that will be discussed in detail in later chapters. Very few
enzymes have actually been exhaustively investigated at all five stages.
4
of catalysis; and (c) understand how an enzyme’s regulatory
interactions affect the catalytic reaction cycle. Ideally, one
should consider as many reasonable models as possible for
the reaction/process of interest. These rival kinetic models
should be as simple as possible: when stripped down to the
bare essentials, any failure of a model to account for an
experimentally determined property of the system becomes
sufficient justification for outright rejection of that model or
for modifying it to account for other by essential properties/
interactions. Simplicity, precision, and generativity – these
are the inherent virtues of highly effective models.
Simplicity demands that a system’s known properties are
represented by the least number of components and/or
interactions. Precision requires explicit presentation of all
required interactions, thus providing an opportunity to
distinguish testable model-specific characteristics of rival
models. Generativity implies that the model should facilitate hypothesis-driven experimentation to test newly predicted properties in a recursive manner that stimulates new
rounds of experimentation. Put plainly, a model is not worth
much, unless it fosters the formulation of new hypotheses
that spur additional rounds of experimentation.
Modern molecular life scientists have become, for want of
a more appropriate appellation, ‘‘interaction spectroscopists’’
– focusing on the spectrum of interactions of proteins and
enzymes with other proteins, nucleic acids, membranes, and
low molecular-weight metabolites, most often in terms of
location, specificity, affinity, and catalysis. And because
enzymes are Life’s actuators, it should not be surprising
that, whenever a significant problem in the molecular life
sciences reaches a sophisticated level of understanding, an
enzyme is almost invariably involved. Because all kinetic
approaches are fundamentally similar, those gaining
mastery over the topics presented in this book can become
proficient at inventing their own kinetic approaches for
testing their own models. Moreover, because biochemical
principles underlie the entirety of the molecular life
sciences, these strategies should also be useful for investigators seeking to unravel the time-ordered events of highly
complex biotic processes in the fields of molecular and
cell biology, physiology and neuroscience, as well as
microbiology and the plant sciences.
Finally, it is worth noting the distinction between
chemical kinetics and chemical dynamics. Both chemical
kinetics and chemical dynamics allow us to infer properties
of transition states and how reactants gain access to them,
but the approaches are fundamentally different. The former
refers to the reactivity (i.e., reaction rates) and bondmaking/breaking mechanisms of chemical transformations,
whereas the latter refers to the atomic and molecular
motions that influence reactivity and stability. Like all
chemical processes, both depend on energy differences
(e.g., DG for the overall reaction, DEact for each elementary reaction, D3 ¼ hDv for each quantized event, Dw ¼ FDx
for the incremental work, where F is a bond force constant
Enzyme Kinetics
or a mechanically generated force, etc.), space (e.g., positionally defined parameters x, y, z in Cartesian coordinates
or r in fields), and time expressed in seconds. In chemical
kinetics, we analyze the dependence of reaction rate on the
concentrations of reactant(s), and although kinetic isotope
effects depend on the masses of nuclei at or near the reaction
center, we are mainly concerned with electronic rearrangements in molecules, as reflected by the nature of the
chemical bonds within reactants, intermediates, and products. (Reaction rate is defined by the product of a reaction
rate constant and its reactant concentration(s), and for
stochastic kinetic approaches, probabilities are often used in
place of macroscopic variables.) In chemical dynamics, the
main goal is to depict how the potential energy changes as
one varies the relative coordinates and momenta of the
atomic nuclei involved in the reaction (Polanyi and
Schreiber, 1974). The latter most often entails the application of classical scattering theory relying on classical
collision theory, with solution of the appropriate equations
for atomic and molecular motions as reactants proceed
along a trajectory on the potential energy surface. At the
single molecule level, population-averaged parameters X
give way to probabilistic expectations <X>, with most
events inevitably stochastic. (Under highly favorable
conditions, one may also pursue quantum mechanical
solutions by solving the appertaining Schrödinger equation(s) for solutions to the appropriate wave function, but
these approaches are only rarely applicable to enzyme
processes, and even then are limited to a small number of
atoms.) Experimental chemical dynamics is most often
pursued in crossed molecular beam experiments, where
each type of reactant molecules, say A or B, is accelerated
within its own beam of molecules to attain a certain energy,
and their reaction (A þ B / C) occurs only where the
beams intersect in an otherwise ultrahigh vacuum that
excludes reactions and interactions with other components.
Changes in chemical composition are then analyzed by
state-resolved spectroscopic techniques. Experiments on
enzymes, however, must be conducted in solution and can
never be analyzed rigorously in the absence of water
molecules. Therefore, the most popular methods for treating
the quantum mechanical sub-systems for enzyme-catalyzed
reactions have been semi-empirical molecular orbital
methods. Alternatively, one may use quantum and classical
dynamics to account for electronic and nuclear effects to
glimpse the time-dependent motion (trajectory) of atoms
within the enzyme and reactant as the solvated enzymebound substrate is transformed into product. Of course,
chemistry and physics are convergent disciplines, and as
computational power expands, enzyme kinetics and enzyme
dynamics will likewise ultimately converge. Again, rather
than settling for the population-averaged properties, enzymatic processes will no longer need to obey simple differential equations, smoothly and deterministically, as defined
by classical chemical kinetics, and we will instead be in
Chapter j 1 An Introduction to Enzyme Science
a position to consider the detailed stochastic behavior of
individual or small ensembles of enzyme molecules.
1.1 CATALYSIS
Only fifty years ago, the most reliable way to estimate the
technological status of a country was to obtain an accurate
estimate of its annual output of sulfuric acid and chlorine
gas or the annual gross production tonnage of aluminum or
steel, especially stainless steel. In this post-industrial era,
the types and amounts of catalysts produced and/or used are
apt to be far more trustworthy indices of economically
advanced countries. Surprisingly, ~20–30% of the Gross
National Product of a so-called first world country depends
in one way or another on catalysis – from cracking of
hydrocarbons, to the synthesis of ammonia and countless
organic molecules, to the formation of high-fructose corn
syrup, and extending to biotechnologies, depending on
enzymes for producing and expressing recombinant DNA,
as well as in stereoselective drug synthesis. Likewise, by reoxidizing auto emissions, in-line catalytic converters reduce
nitrogen oxide pollutants from internal combustion engines.
Catalysis is a mainstay of any modern economy, and
products of catalysis play essential roles in our everyday
life – from the petrochemistry and agrochemistry to medicine and nutrition. As endo- and exo-cellulases become the
mainstay for ethanol production from an ever-widening
range of cellulosic sources, enzyme catalysis will take on
even greater significance in biofuel production. It is likely
that a country’s GNP will soon be as inextricably linked to
its enzyme technology as to its gold supply.
1.1.1 Roots of Catalysis in the Earliest
Chemical Sciences
Exactly when humans first became aware of catalysis will
always remain a mystery, but its effects were manifestly
significant to hominids. Through trial-and-error and a keen
perception, the ancients discovered a variety of substances
that accelerate or retard chemical reactions. By nurturing an
already glowing ember as a primitive oxidative surface
catalyst, they learned how to harness combustion. Later, they
mastered the use of friction to create their own embers, and
igniting new fires at will. Long before its first mention in the
Iliad, herders had observed that the contents of goat and
sheep stomachs curdled milk, thus discovering a key enzymatic reaction that greatly facilitated cheese production.
They likewise learned to dehydrate and stabilize foodstuffs
through salting and smoking – unwittingly inhibiting
hydrolases and deactivating oxidases. The early Egyptians
likewise mastered the fine art of mummification, again by
inhibiting digestive and oxidative enzymes. Humans also
found that strong alkali hastened saponification of tallow, and
the art of soap making was born. Others observed that the
5
presence of certain metal ions promoted vinegar formation.
Archeological evidence for their pervasive use suggests that
early humans recognized and prized these catalyst-based
technologies long before the existence of a written record.
The Russian chemist Gottlieb Kirchhoff in 1812 is
credited as the first to document the enhanced rate of glucose
formation from starch in the presence of various acids. The
English chemist Humphry Davy likewise observed that many
gases burned more vigorously in the presence of metallic
platinum, and his Irish namesake Edmund Davy was the first
to discover a spongy form of platinum with remarkable gas
absorptive and catalytic properties. Yet, it was the Swedish
chemist Jons Jacob Berzelius, whose studies of diastase,
a crude preparation of a-amylase, unified these and other
observations with the germinal concept that, to hasten
product formation, a catalyst must first combine with its
reactant(s). In his extraordinary writings, Berzelius combined
the Greek words kata and lyein to coin the term catalyst as
any agent that promotes chemical reactivity by first
combining with a reactant to weaken its stabilizing bonds. In
the translation of Jorpes (1966), Berzelius said that the word
‘‘catalyst’’ denotes ‘‘substances that are able to awaken
affinities that are asleep at one temperature by their mere
presence and not their affinity.’’ The former property
implicitly anticipates the catalyst’s ability to lower a reaction’s activation energy, with the latter suggesting that the
equilibrium poise should be unaffected. While others had
suggested that catalysts acted at a distance, Berzelius
correctly inferred that catalytic action required complexation
of catalyst and reactant. Through the examination of the
thermal decomposition of HI into H2 and I2, the French
chemist Lemoine also suggested that, while the presence of
metallic platinum accelerated the reaction, the catalyst is
without effect on the reaction’s final equilibrium position.
Kinetic experiments proved to be indispensable in
efforts to define many fundamental chemical principles.
Ludwig Wilhemy (1850), for example, used polarimetry to
quantify the rate of acid-catalyzed hydrolysis of sucrose4
4
In this reaction, the dextrarotatory reactant sucrose (specific rotation ¼
þ66.5 ) is converted to an overall levorotary product mixture, owing to
the fact that for D-glucose equals þ52.7 and that for D-fructose equals
92.4 . The hydrolysis of sucrose therefore yields a net leftward
rotation of 39.7 . Because the state of polarization ‘‘inverts’’ (i.e.,
changes from a (þ) to a () rotation), the enzyme catalyzing this
hydrolysis of sucrose into D-glucose and D-fructose was accorded the
name invertase. Prior to the advent of photomultiplier tubes and stable
electronic circuitry, polarimetry offered a simple and reliable
quantitative way of assessing the concentration of optically active
substances as well as those optically inactive compounds that generate
optically active product(s). To assess concentration, chemists of that era
also used split-field optical comparitors, relying on the naked eye to
assess the color intensity of an experimental solution relative to that of
a solution of known concentration. In addition to being less accurate
and far less sensitive than polarimeters, comparitors proved to be far
more susceptible to experimental bias.
6
to show that the rate of this reaction is linearly dependent
on the concentration of sugar. Berthelot (1862) and
Berthelot and de Saint-Gilles (1862) reached the same
conclusion from studies on ethyl acetate hydrolysis, and
such observations led Guldberg and Waage (1867; 1979) to
postulate that chemical reactions must be highly dynamic,
with reactants and products relentlessly interconverting
into each other, even at equilibrium. In advancing this
principle, widely known as the Law of Mass Action, they
suggested that the rate in each direction of a reversible
reaction depends on reactant concentration (often
expressed as the intensive variable molarity) and not the
amount of substance (commonly given by the extensive
variable mole).
As discussed at length in Chapter 3, the modern
conceptual framework for the discipline known as
chemical kinetics was founded late in the nineteenth
century by the powerfully insightful contributions of the
Swedish chemist Svante Arrhenius and the German
chemist Jacob van’t Hoff, who both became Nobel
Laureates in chemistry. They and German physical
chemist Wilhelm Ostwald, the Nobelist credited for first
expressing reaction velocity as a change in reactant
concentration per unit time (i.e., v ¼ d[Reactant]/dt),
established the enduring concept that catalysts promote
reactivity without altering the equilibrium position of the
overall chemical reaction. These investigators recognized
that thermodynamics constrains catalysis: after each
catalytic round, the catalyst releases its product and
therefore cannot exert any cumulative effect on the
reaction’s standard Gibbs free energy change DG . This
discovery increased the determination of chemists to
discover catalytic substances and even to design artificial
catalysts endowed with special properties. Speed and
yield are the essence of catalysis, but the idea that one
may impart reactivity to otherwise unreactive substances
lies at the heart of modern chemical enterprises. Nowhere
is this more evident than in the work of Fritz Haber, the
notorious German chemical engineer5 and Nobel
Laureate. Haber’s research team overcame the virtual
inertness of dinitrogen by carrying out some 20,000
experiments, utilizing thousands of catalyst preparations
under a wide range of reaction conditions. They eventually settled on the use of iron filings to catalyze ammonia
synthesis from N2 and H2 at high temperature (600–800
K) and extreme pressure (300 atm). High temperature
facilitated dissociation of highly stable bonds within N2
and H2, and pressure displaced the reaction equilibrium in
5
During World War I, Haber supervised firsthand battlefield tests on the
efficacy of chemical warfare agents that later proved to irreversibly
inhibit the enzyme acetylcholine esterase. Such activities would be
subject to prosecution under the international treaties on war crimes
signed at the close of that war.
Enzyme Kinetics
favor of ammonia (Reaction: N2 þ 3H2 ¼ 2 NH3). That
process – now bearing Haber’s name – has forever altered
the human condition by augmenting Nature’s output of
‘‘fixed’’ nitrogen by some 20–30%. To improve crop
yields, farmers routinely inject synthetic ammonia
directly into the soil.
In considering the nature of a catalytic cycle, one may
take the case of heterogeneous catalytic decomposition of
the toxic atmospheric pollutant N2O within the catalytic
converter of a modern automobile. The cycle begins with
chemisorption of N2O onto the platinum/palladium catalyst, a step that weakens the bonds stabilizing nitric oxide,
to the effect that the N–O bond can dissociate. The latter
produces N2, which desorbs from the surface, leaving
oxygen radicals on the catalyst. At the catalyst’s operating
temperature, these radicals diffuse along the metallic
surface until two of them encounter each other and
combine to form O2, the latter then desorbing from the
surface. French Chemist Paul Sabatier (Nobel Laureate in
1912) is credited with a principle bearing his name. Stated
in its simplest form, the Sabatier Principle asserts that for
effective catalysis, substrates and products must bind
sufficiently tightly, so as to promote catalysis, but not too
tightly so to prevent catalysis. Sabatier stressed the
momentary nature of catalytic intermediates, a point that
underscores their celerity and the importance of kinetics in
analyzing their nature.
As discussed below, catalytic selectivity/specificity
also allows chemists to control the stereochemical
outcome of reactions that would be otherwise nonspecific. And while organic chemistry of the 1950s relied on
just a few catalysts (mainly Hþ, OH, Al3þ, Fe3þ, as
well as elemental Pt, Ni, and Pd) that were almost
invariably stereochemically unselective, modern organic
chemists have exploited a much wider repertoire of
metallo-catalysts.
Recognizing that all reactions proceed through the
formation and turnover of transition-state intermediate(s), one may consider the conversion of reactant A
into product P in the absence and presence of catalyst C.
In the uncatalyzed case, reactant A isomerizes through
a succession of intermediates and transiently reaches the
activated complex Xz. As the least stable intermediate,
Xz exhibits an equal likelihood of reconverting to the
reactant or going onward to product, such that the system
eventually reaches thermodynamic equilibrium. In the
catalyzed reaction, reactant A first combines with catalyst C to form the C$A, which then passes through
a series of intermediates (e.g., C$X1, C$X2, etc.) to reach
C$Xz. As was true for the uncatalyzed process, the
intermediate C$Xz can either return to C$A or advance to
C$P, with product-release subsequently regenerating the
catalyst. Michael Polanyi, father of Nobel chemistry
laureate John Polanyi, was arguably the first to articulate
the notion that stabilization of the reaction transition-state
Chapter j 1 An Introduction to Enzyme Science
7
Xz as the complex of catalyst and transition-state C$Xz
should greatly increase the forward and reverse reaction
rates. The enhancement factor 3 (equal to vcat/vuncat)
therefore applies both to the forward and reverse reactions, and a reaction’s equilibrium constant can be
expressed as:
K ¼
3 vuncatalyzed
vuncatalyzed
¼ reverse
¼ Keq
3 vreverse
vuncatalyzed
uncatalyzed
1.1
Autocatalysis is a special case of chemical catalysis in
which the active catalyst is also a product. An example is the
formation of pepsin on its storage form pepsinogen in acidic
gastric juices:
Initiating Reaction:
Pepsinogeninact + H+ → Pepsinact
Autocatalytic Reaction: Pepsinogeninact + Pepsinact → 2 Pepsinact
Scheme 1.2
where the inactive zymogen Pepsinogeninact is at first
converted slowly by acid catalysis to the active enzyme
Pepsinact, it then rapidly catalyzes the conversion of
any remaining zymogen to its active form. Note that
each catalytic round during the autocatalytic phase
doubles the amount of active enzyme until the concentration of inactive enzyme is depleted (see Section 3.9.4:
Autocatalysis).
Finally, the catalyst concentrations approach the
concentrations of substrate(s) or product(s), the equilibrium position of the reaction, depending on the catalyst’s
relative affinity for the reactant(s) and product(s). This
effect can manifest itself in some rapid-mixing experiments, particularly when reagent concentrations of enzyme
are utilized.
1.1.2 Synthetic Catalysts
Chemists have created powerful catalysts that facilitate
chemical transformations in chemistry laboratories, oil
refineries, and even automotive exhaust systems. Table 1.1
summarizes some of the most widely used catalysts that
contribute to the trillion dollar petrochemical and agrichemical industry worldwide. Although metallic platinum,
palladium and nickel are constituents in many catalysts, the
active forms consist of small surface imperfections, or step
defects, and not merely the projected geometry of the metal’s
internal crystal surface. These agents are often called
heterogeneous catalysts, a term that indicates the presence of
two phases: gaseous or liquid reactants binding and reacting
on the surface of a solid catalyst. Catalysts, such as hydroxide
ions and protons, which remain in the same phase as the
reactants, are referred to as homogeneous catalysts.
Synthetic catalysts are often highly stable, allowing
them to operate efficiently even in the face of elevated
temperatures and pressures, as well as extremes of pH.
One unrelenting problem has been to design catalysts that
resist fouling, or quenching, by tight-binding reaction
products and/or metal ions. Most synthetic catalysts are
also inferior to biological catalysts in at least four other
respects, as they: (1) are less efficient at physiologic
temperature, low pressure, and neutral pH; (2) are relatively unselective; (3) rarely display sufficiently high
chiral recognition, a property that greatly limits their use
in preparing optically active biomolecules; and (4) are not
regulated by feedback activators and/or inhibitors.
1.1.2a Catalytic Hydrogenation
The classical case of catalytic hydrogenation (Fig. 1.2) is
a two-phase, or heterogeneous, process. The alkene or
alkyne is first adsorbed on the surface of the catalyst
alongside a dihydrogen molecule, whereupon the catalyst
TABLE 1.1 Selected Man-made Catalysts and the Reactions Catalyzed
Catalyst
Category
Process/Properties
Platinum-containing Chlorinated Alumina
Heterogeneous
Platinum, Nickel, Palladium
Iron Shavings/Dust
Silica/Alumina Zeolites; NiCoMo tri-metallics
Vanadium(V) Oxide, Palladium
Acids/Bases
Heterogeneous
Heterogeneous
Heterogeneous
Heterogeneous
Heterogeneous/
Homogeneous
Heterogeneous
Homogeneous
Homogeneous
Hydroisomerization (conversion of n-butane into
isobutane)
Hydrogenation of double bonds
Haber ammonia process (Reaction: 3 H2 þ N2 # 2 NH3)
Cracking of petroleum into volatile fuels
Oxidation of exhaust from internal combustion engines
Hydrolysis of carboxylic/phosphoric esters and anhydrides
Grubbs and Hoyeyda Ruthenium Catalysts
Chiral Catalysts
Catalytic Antibodies
Metathesis (see text for details)
Enantiomeric selectivity/specificity
Over 100 different reaction types
Enzyme Kinetics
8
H
H
H
H
C
C
H
H
FIGURE 1.2 Schematic of catalytic hydrogenation of ethylene on
a nickel, platinum, or palladium surface. In this idealized representation, the metal surface acts as a rack, on which each of the reactants is
stretched by binding to adjacent metal atoms. This physisorptive process
occurs by interactions of reactant electrons with empty electron-deficient
orbitals of the metal. Catalytic hydrogenation results from the heightened
reactivity among the weakened intramolecular bonds of H–H and
CH2]CH2, depicted above as dashed lines between reactant atoms. In
many metals, step-like dislocations on the crystal surface are the actual
sites of enhanced catalytic action. Except for the fact that heterogeneous
catalysis occurs at the interface of a gas-solid, liquid-solid, or immiscible
liquid-liquid phases, the process of catalytic hydrogenation resembles
a random bisubstrate enzyme-catalyzed reaction (i.e., reactants A and B
add randomly to form an Enz$A$B, followed by conversion to product
complex Enz$C, from which C desorbs from the active site to complete
the catalytic cycle).
weakens their respective bonds and may even change the
position and/or orientation of these bound species. The two
hydrogen atoms then shift from their interactions with the
metal surface to the carbon atoms comprising a double or
triple bond, with attendant formation of a more saturated
hydrocarbon. The latter is more weakly adsorbed and soon
departs from the catalyst’s surface. The exact nature and
timing of these events is still incompletely understood.
What is clear is that the metal surface acts as a rigid rack on
which the reactants are stretched to weaken the s-bond of
H–H as well as the p-bond of an alkene (or alkyne), with the
effect that hydrogenation is facilitated. Because H–H and
R–C]C–R9 (or R–R9) bond lengths differ by ~0.5 Å, each
bond must be polarized to a different degree to reach the
optimal reaction transition-state. (A corollary is that the
likelihood of achieving this alignment in the catalyst’s
absence is extraordinarily low.)
Classical transition metal catalysts, such as platinum,
palladium, nickel and rhodium, rely on their intrinsic
inter-atomic spacing in their crystalline state or as multiatom aggregates. That said, some catalysts actually rely
on step-dislocations on their roughened surfaces to create
the best sites for catalytic hydrogenation. To achieve
higher catalytic rate enhancements, many synthetic catalysts are deliberately designed to contain reactive surface
defects or atomic dislocations. No metal surface is
perfectly flat, and catalysis may be more effective in
surface microenvironments. Another useful strategy is to
deposit metal atoms onto other solid substrates that can
greatly influence the resulting surface geometry and
coverage.
Given the great cost of metals like platinum, palladium
and even nickel, chemists have attempted to maximize
the active catalytic surface of metal catalysts. In such
cases, platinum and palladium are combined with a charcoal support (also called the substratum or substrate).
Raney nickel, for example, is a solid hydrogenation
catalyst composed of fine grains of a nickel-aluminum
alloy, and the catalyst known as ‘‘platinum on charcoal’’
consists of 5% platinum and 95% charcoal by weight.
Gold nanoparticles have also been employed as catalysts.
To explain why ordinarily inert gold becomes a powerful
catalyst, chemists have proposed that: (a) nanometersized particles contain many more surface dislocations
that serve as unusually reactive domains; (b) they are
more or less electron dense than bulk gold; (c) such
particles contain numerous perimeter sites and/or
‘‘sticky’’ paracrystalline surfaces, and/or (d) nanometer
sized particles have different metallic properties than
those of bulk gold (Bell, 2003). Other industrial catalysts
include di- and poly-nuclear metal cluster complexes,
such as di-molybdenum and di-tungsten complexes, dirhodium (II) complexes, as well as multinuclear Rh, RhCo, and Ir-Co complexes.
1.1.2b Metathesis
The process known as olefin metathesis refers to position
changing organochemical catalysis occurring in the
presence of suitable transition metal complexes, including
various metal carbenes. These catalysts (particularly the
Grubbs Ruthenium Catalyst and Hoveyda Ruthenium
Catalyst) facilitate bond-breaking and exchange of
substituents directly attached to the double bonds of the
coordinated olefins. A metal carbene initiates olefin
metathesis by reacting with an olefin to form a metallated-cyclobutane intermediate, which then breaks apart
to form a new olefin and a new metal carbene. This highly
versatile chemical process can be used to: (a) swap
groups between two acyclic olefins (a process called
cross-metathesis); (b) close large rings (ring-closing
metathesis); (c) form dienes from cyclic and acyclic
olefins (ring-opening metathesis); (d) polymerize cyclic
olefins (ring-opening metathesis polymerization); and (e)
polymerize acyclic dienes (acyclic diene metathesis
polymerization).
The commercial availability of these catalysts has
greatly promoted the use of metathesis in macrolide
synthesis, where closure of large rings (i.e., those having
ten or more atoms within them) is typically a low-yield
reaction. The power of olefin metathesis is that it
Chapter j 1 An Introduction to Enzyme Science
9
transforms the –C]C– double bond, a functional group
that is often unreactive toward many reagents. With certain
catalysts, new –C]C– double bonds are formed at or near
room temperature, even in aqueous media using starting
materials that bear a variety of functional groups. Chemists
Yves Chauvin, Robert Grubbs and Richard Schrock shared
the 2005 Nobel Prize in chemistry ‘‘for the development of
the metathesis method in organic synthesis.’’ That these
processes are highly relevant to the synthesis of enzyme
inhibitors and therapeutic agents is illustrated by the use of
tandem ring-closing metathesis to and subsequent hydrogenation to synthesis conformationally restricted cyclic
dinucleotides joined with saturated connections between
the nucleobase and the phosphate moieties (Borsting and
Nielsen, 2002). Metathesis also holds great promise for
industrial-scale reactions that are environmentally compatible (so-called Green Chemistry). One concern, however,
is the high cost, currently around $100 per millimol catalyst. Another is the use of toxic ruthenium, molybdenum,
tantalum, etc. A third concern is the relative inefficiency of
these catalysts, which typically operate at concentrations
that are 210 mol-% of the reactant concentrations.
H3C CH3
H3C
O
H3C
O
*
C
*
*C
*
O
O
OH
OH
*
H3C
N
N
*
C
C
CH3 H3C
H3C
CH3
CH3
bis(Oxazoline)
TADDOLate
**
H
N
O
H
N
O
Co
O
O
O
O
t-Bu
t-Bu
t-Bu
t-Bu
O
O
O
O
Co
O
N
H
O
N
**
H
1.1.2c Chiral Catalysts
To mimic the remarkable enantiomeric preference exhibited by many enzymes toward their chiral substrates,
chemists have struggled to design homogeneous catalysts
that are enantioselective. The trick is to introduce the right
mix of binding energy, functional group chemistry, and one
or more chiral and/or dissymmetric sites (Yoon and
Jacobsen, 2003).
One such catalyst, known simply as TADDOLate
ligand, is based on the structure of tartaric acid,6 one of the
least expensive, naturally occurring chiral substances.
(Asymmetric carbon atoms are indicated by asterisks.)
TADDOL catalyzes aldehyde alkylation, ester acoholysis,
and iodo-lactonization. Another Diels-Alder catalyst
Salen Complex
Bis(oxazoline) is loosely based on the structure of vitamin
B12. Metal ion-containing catalysts, known as Salen
complexes, facilitate epoxidation, epoxide ring-opening,
imine cyanation, and conjugate addition reactions. Such
compounds combine with reactive metal centers to produce
catalysts that effectively create asymmetric environments
that promote the selective binding and/or enhanced reactivity. As pointed out by Yoon and Jacobsen (2003), exactly
what structural features account for the broad applicability of
synthetic chiral catalysts remains unclear. They suggest these
6
Tartrate enjoys the distinction as the substance that Pasteur used to formulate his germinal ideas about stereochemistry as well as the first to have its
absolute stereochemical configuration determined (Bijvoet, Peerdeman, and van Bommel, 1951). By verifying Emil Fischer’s fortuitous assignment
for (þ)-glyceraldehyde (Rosanoff, 1906), there was no need to revise existing chemistry textbooks.
COOH
COOH
H
HO
OH
H
H
C
OH
HO
C
H
COOH
HOOC
(R)
H
COOH
OH
H
(R)
CH3
OH
H
OH
H
OH
COOH
COOH
Fischer Projection
Fischer Projection
Cahn-Ingold-Prelog
Newman Projection
Shown above are various equivalent projections depicting the absolute stereochemical configuration of (þ)-tartrate. It is also worth noting that Pasteur
(1858) reported the first stereospecific enzyme-catalyzed reaction, in which yeast fermented dextrarotatory tartaric acid, while leaving levorotatory
tartaric acid completely intact.
Enzyme Kinetics
10
catalysts possess rigid structures with multiple oxygen,
nitrogen, and phosphorus atoms that allow them to interact
strongly with reactive metal centers. Because these agents
have a two-fold axis of symmetry, the number of possible
transition-state geometries is likely to be more limited.
An inherent limitation in the design of chiral catalysts is the
current inability of chemists to reliably predict the type of
reactions that will be facilitated by a particular agent, the
extent of its stereoselectivity, or the achievable catalytic rate
enhancement. For example, titanium complexes of chiral
peptide-based Schiff’s base (or imine) ligands catalyze
cyanation of epoxides, aldehydes, and imines with high
enantioselectivity; the corresponding copper complexes
catalyze allylic substitution of dialkyl-zinc nucleophiles;
whereas analogous zirconium complexes catalyze dialkylzinc addition to imines (Josephson et al., 2001). Absent
a predictable outcome, one is left with the unenviable task of
surveying the reaction spectrum of each newly prepared
synthetic catalyst. The advent of High-Throughput Screening
(HTS) promises to lessen the load of determining a catalyst’s
reactivity profile, but this approach remains to be perfected.
1.1.2d Catalytic Antibodies
Among ‘‘semi-synthetic’’ catalysts listed in Table 1.1 are
catalytic antibodies (also known as abzymes). These bioengineered proteins can be designed to accelerate specific
organic chemical reactions. Basing his ideas on assertions
about transition-state stabilization (Haldane, 1930; Pauling,
1946; Evans and Polanyi, 1936), Jencks (1969) succinctly
advanced the following argument for catalytic antibodies:
If complementarity between the active site and the transition state contributes significantly to enzymatic catalysis,
it should be possible to synthesize an enzyme by constructing such an active site. One way to do this is to prepare an
antibody to a haptenic group, which resembles the transition state of a given reaction. The combining sites of
such antibodies should be complementary to the transition
state and should cause an acceleration by forcing bound
substrates to resemble the transition state.
Because transition states are intrinsically unstable,
catalytic antibodies are selected by using chemically stable
transition-state analogues used as immunogens. For
example, antibodies generated against a bent porphyrin ring
were found to catalyze the metallation of heme groups,
presumably by straining the planar substrate toward a bent
transition-state conformation.
In the classical ‘‘Bait-and-Switch’’ approach, one designs
a hapten (i.e., an immunogenic molecule that serves as the
‘‘bait’’) that structurally resembles a likely transition-state
species (Pollack, Jacobs and Schultz, 1986; Tramontano,
Janda and Lerner, 1986). In selecting the best haptens, one
focuses on key features of the transition-state intermediate,
such as the arrangement of its atoms and/or its electrostatic
charge. For example, were one interested in producing an
antibody with the activity of a glycosidase, one might chose
a modified sugar that resembles the oxa-carbenium ion
intermediate with a half-chair conformation at or near the
glycosyl carbon atom (see nucleoside hydrolase mechanism
in Section 8.12.4; or lysozyme mechanism in Section 9.8.5c).
The desired outcome is that the chosen hapten elicits antibodies that, when switched to bind on substrates, have the
capacity to facilitate the desired reaction. The candidate
hapten is then coupled to a protein carrier, typically keyhole
limpet hemocyanin (KLH), and the resulting conjugate is
used to immunize mice to produce one or more monoclonal
antibodies. Catalytic antibodies are then identified on the
basis of their ability to catalyze the reaction of interest when
exposed to the desired substrate instead of the hapten (i.e., is
‘‘switched’’). Because catalytic antibodies may not attain the
same stereospecificity as natural enzymes, and because
catalytic antibodies may catalyze side-reactions, the experimenter is well advised to characterize the products with
respect to structure and enantiomeric purity.
In the case of ester hydrolysis, a phosphonate is
a reasonably good isostere of the enzyme’s tetrahedral
oxyanion transition state, with specificity determined in part
by the side chains R and R9.
R'
R'
OH
O
R
O
O
C
O
Transition-State
Structure
R
P
O
Transition-State
Analogue
One then raises monoclonal antibodies against the phosphonate-modified keyhole limpet hemocyanin (KLH). Alternatively, one may select bacteria that express catalytic Fab’s
(i.e., antigen-binding fragments of antibodies) that are generated by recombinant DNA methodology. Each antibody is then
isolated and then evaluated for its ability to catalyze the
hydrolysis reaction of interest. Observed rate enhancements
should correlate with an antibody’s affinity for transition-state
analogue (TSA) versus reactant (R) (i.e., KTSA/KR, where KTSA
¼ [Ab$TSA]/[Ab][TSA] and KR ¼ [Ab$R]/[Ab][R]). Experimental results, however, often fail to satisfy the simplistic
assumption that the more closely an analogue resembles
a reaction transition state, the more effective is the antibody as
a catalyst.
One inherent limitation in the use of transition-state
analogues to generate catalytically proficient antibodies is that
many interesting enzyme reactions are inevitably multi-step
reactions, each with its own transition states. Therefore, no
single analogue is likely to be an adequate template for each
transition state. A second factor limiting the catalytic efficiency of catalytic antibodies is the relative inflexibility of
most antibodies. While most enzymes are highly flexible and
Chapter j 1 An Introduction to Enzyme Science
11
contain few internal disulfide bonds, the opposite is true of
antibodies. A third limitation is that there is no easy way to
increase the rate of product release in the design of catalytic
antibodies. For most enzyme-catalyzed reactions, chemical
interconversion of enzyme-bound substrate and enzymebound product is fast, and product release is frequently the
rate-limiting step. The observed rate enhancements for
enzyme-catalyzed reactions therefore most often measure the
rates of product release. So increasing the rate of chemical
interconversion of an antibody-bound substrate and antibodybound product may not do much to improve the observed rate
enhancements for antibody-catalyzed reactions.
Reactive immunization is a new procedure for generating
catalytic antibodies that tackles this problem by employing an
antigen that is so highly reactive that a chemical reaction occurs
in the antibody-combining site during immunization (Wirsching
et al., 1995). In the initial application of this approach, an
organophosphorus diester hapten was used as a ‘‘reactive
immunogen.’’ A large number of the resulting antibodies catalyzed the formation and cleavage of phosphorylated intermediates and subsequent ester hydrolysis. Wagner, Lerner and Barbas
(1995) applied the reactive immunization technique to generate
antibodies that catalyze the aldol reaction. The mechanism for
antibody catalysis of this reaction mimics that used by natural
Class-I aldolase enzymes. Immunization with a reactive
compound covalently trapped a Lys residue in the binding pocket
of the antibody by formation of a stable vinylogous amide. The
reaction mechanism for the formation of the covalent antibodyhapten complex was recruited to catalyze the aldol reaction. The
antibodies use the epsilon-amino group of Lys to form an
enamine with ketone substrates and use this enamine as a nascent
carbon nucleophile to attack the second substrate, an aldehyde, to
form a new carbon–carbon bond. Barbas et al. (1997) later
designed additional antibody catalysts for aldol condensation
reaction, based on the intermediates shown in Scheme 1.3.
O
O
H
Enz
R1
N
CH3
HO
CH3
R3
R2
O
Substrate
O
Enz
HN
R1
O
CH3
R3
H
R2
O
Intermediates
Product
Scheme 1.3
The observed rate enhancement 3 of 4,000,000 for this
catalytic antibody far exceeds the 103 to 105 values for
others (Barbas et al., 1997), but falls short of aldolase
(Reaction: Fructose-1,6 Bisphosphate # Glyceraldehyde3-P þ Dihydroxyacetone-P) by 8 to 10 orders of magnitude.
A fortuitous case of an engineered antibody catalyzing
a multi-stage transesterification reaction was reported by
Wirsching et al. (1995). This antibody behaved as a Ping Pong
enzyme (Catalytic Reactions: E þ S1 # E$S1; E$S1 # F þ P1;
F þ S2 # F$S2; F$S2 # E þ P2, where E and F are the free
enzyme and the acyl-enzyme, respectively). Evidence for multistage catalysis was adduced by the parallel-line patterns observed
in a plot of 1/v versus [Ester] at several constant levels of the acylacceptor alcohol (AAA) and in a plot of 1/v versus [AAA] at
several constant levels of the ester. The resulting steady state
kinetic parameters were 3 and 7.3 mM, respectively, for the ester
and alcohol, and kcat was 21 min1 (the latter obviously much
slower that natural enzyme counterparts). The authors found that,
when a structurally related p-nitrophenyl ester was added to
varying concentrations of the antibody with rapid mixing, equimolar amounts of p-nitrophenol formed quickly, followed by
a slower, steady-state release phase. The amplitude of the burstphase was proportional to the catalyst concentration.
Other semi-synthetic enzymes have been prepared by
modifying binding proteins and enzymes. For example, Zemal
(1987) observed catalysis of p-nitrophenylester hydrolysis
(enhancement factor 3 ¼ 1900) by heme-depleted myoglobin,
a property that can be explained by the apolar binding pocket
with its two imidazoles that normally interacts with the heme.
Likewise, upon attachment of a flavin cofactor to Cys-25
within papain’s active site, the resulting synthetic enzyme (or
synzyme) was found to catalyze oxidation of dihydronicotinamide to nicotinamide with concomitant reduction
of the flavin (Kaiser and Lawrence, 1984; Slama et al., 1984).
What becomes clear from model studies is that enzymes do
much more than stabilize reaction transition states: they bind,
orient, desolvate, and destabilize substrates; they push/pull
protons to/from substrates, intermediates and products; they
promote nucleophilic reactivity; and they exploit metal ions as
templates, as Lewis acids, and as highly reactive redox species
that are otherwise inaccessible in aqueous medium. Enzymes
also exhibit a remarkable capacity to manage enthalpy and
entropy changes throughout the catalytic reaction cycle,
culminating in the release of reaction products. Although the
most up-to-date approach uses a transition-state analogue to
generate the initial specificity, followed by site-directed
mutagenesis to provide essential catalytic groups, obtaining
catalytic antibodies is still hit-or-miss.
Underscoring the limited rate enhancements achieved with
catalytic antibodies is the discovery that a so-called off-the-shelf
protein (bovine serum albumin) exhibits rate enhancements that
rival tailor-made catalytic antibodies. Noting that Thorn et al.
(1995) described an antibody catalyzing the eliminative ringopening of benzisoxazole, Hollfelder, Kirby and Tawfik (2001)
tested whether the lysine side-chain amines might also participate
Enzyme Kinetics
12
in this general base-catalyzed reaction. With human albumin,
they obtained a kcat of 28.8 9.7 min1, albeit with a prompt
onset of product inhibition after only around 10 catalytic cycles.
They also found that the rate enhancements reported for catalytic
antibodies depended on the somewhat arbitrary choice of solvent
conditions applied to the reference reaction. Until chemists can
increase the flexibility of catalytic antibodies, the ability to
‘‘teach’’synthetic catalysts and antibodies to mimic enzymes will
remain an insuperable task.
1.1.2e Synthetic Enzymes
Enzyme chemists have labored assiduously to fashion novel
catalysts from structural proteins or to transform biospecific
ligand binding sites into active sites. Although the development of crown ethers by Nobelist Donald Cram is often
erroneously credited as an early breakthrough in the synthesis
of artificial enzymes, U.S. chemists Myron Bender and Ronald
Breslow pioneered these efforts. Bender and Breslow used
synthetic organic chemistry to introduce catalytically active
substituents (e.g., chiefly carboxyl and imidazole groups) on
the rim of cavity containing cyclodextrins (see Section 7.11 for
a discussion of cyclodextrin inclusion complexes). Before his
early demise, the American chemist E. Thomas Kaiser had
attempted to refashion the active sites of various heme proteins
and a few enzymes to create synthetic enzymes with novel
catalytic properties. His creative efforts were met with modest
progress toward the goal of fashioning new biocatalysts. The
monograph Artificial Enzymes edited by Breslow (2005)
presents a series of cogent reviews on artificial enzymes,
including biomimetic chemistry, vitamin B6-based enzyme
models, synthetic polymers with enzymatic activity, catalytic
antibodies, protein-based artificial enzymes, artificial metalloenzymes, as well as artificial restriction enzymes. To date,
these efforts have met with uninspiring success, often for the
same reasons already noted above for catalytic antibodies.
1.2 BIOLOGICAL CATALYSIS
That the rates of enzyme-catalyzed reactions7 were studied
long before corresponding organic chemical reactions
7
It is helpful to understand some basic terminology used by enzymologists.
A simple enzyme is a biological catalyst made wholly of protein, although
more than one polypeptide chain may be part of the active enzyme. A
complex enzyme is composed of one or more polypeptide chains plus
a low-molecular-weight organic molecule or metal ion at its active site.
The term holoenzyme refers to the entire complex enzyme, whereas the
term apoenzyme refers only to the protein component. If the non-protein
component binds non-covalently to the apoenzyme, it is called
a coenzyme. (Many coenzymes contain structural elements of vitamins.) A
metal ion that binds directly to the protein is called a metal ion cofactor.
A prosthetic group is a relatively small organic molecule that is usually
extremely tightly, or even covalently, bound. Some prosthetic groups also
contain a metal ion (e.g., heme is protoporphyrin IX plus Fe(II) or Fe(III)
ion) held in place by coordinate covalent bonds.
shouldn’t be surprising. While Fritz Wöhler had succeeded
in synthesizing urea in 1826, the field of physical organic
chemistry, which deals with the underlying kinetics and
mechanisms of organochemical reactions, developed relatively slowly until the late nineteenth century. What most
limited the progress in chemical kinetics of organic and
inorganic reactions was the lack of reliable methods for
quantifying changes in the concentration of reactants or
products. Spectrophotometers were nonexistent, because
the then primitive electronic circuitry and low voltages
available from batteries precluded the fabrication of photomultiplier tubes. The need for a conveniently observable
property led to early studies on the action of a crude
preparation of emulsin on the hydrolysis of emulsified
amygdalin, a sparingly soluble ester isolated from apricot
pits. Emulsin later proved to be an enzyme that readily
converts the visibly milky white, aqueous suspension of
amygdalin into transparent (water-soluble) products. With
that simple assay, the concept of catalysis could be
demonstrated. It was, however, the advent of the polarimeter that made possible the quantitative investigation of
how reaction rate depends on changes in the concentration
of optically active reactants or products. Even so, organic
chemists lacked the means to synthesize chiral compounds,
the latter being the sole province of physiologic chemistry
(biochemistry). Because the degree of rotation of planepolarized light was a linear function of the molar
concentration of an optically active substance, this technique provided the opportunity to demonstrate unambiguously that the acid-catalyzed hydrolysis of sucrose brought
about stereochemical inversion (i.e., a change in the
direction of rotation of plane polarized light. Likewise, the
corresponding action of the enzyme invertase (Reaction:
Sucrose þ H2O # D-Fructose þ D-Glucose) could be
monitored reliably. The availability of polarimetry as
a simple, highly sensitive, and reproducible quantitative
technique essentially established chemical kinetics as
a rigorous physical science.
1.2.1 Roots of Enzyme Science
The origins of enzymology as a scientific discipline can be
traced to Spallanzani who, in 1783, demonstrated that
gastric juices liquefied meat, and to Gay-Lussac who, in
1810, reported that yeast growing anaerobically could
ferment sugars into ethanol and CO2. Enzymes were first
discovered in 1833 when Anselme Payen and Jean Persoz
found that an alcohol precipitate of malt extract contained
the thermolabile substance diastase, which converted starch
into sugar. Justus von Liebig proposed that fermentation and
digestive processes were inherently the result of chemical
action. In 1835, the German physiologist Theodor Schwann
discovered that, in a manner similar to that of acid (as
discovered decades earlier by American physiologist John
Young), gastric juice also contained its own digestive
Chapter j 1 An Introduction to Enzyme Science
substance that became known as pepsin (from the Greek
pepsis for digestion). Work many years later established that
pepsin is an enzyme.
Given his remarkable chemical intuition and role as
a reductionist, it is remarkable that the great French
chemist Louis Pasteur steadfastly adhered to the view that
fermentation was uniquely the province of living yeast
cells. His view supported the vitalists, who asserted that
life is the manifestation of a vital force (or, e´lan vital), the
life-creating principle immanent in all living organisms.
The opposing mechanistic view that living systems would
inevitably be shown to obey the laws of chemistry and
physics was held by the German physiologist William
Kühne, who in 1878 coined the phrase enzyme (from the
Greek en and zyme, standing for ‘‘in’’ and ‘‘yeast’’) for
the fermentative substance in yeast. In 1893, the Latvian
scientist Wilhelm Ostwald formally classified enzymes as
catalysts, even though their chemical nature was still
widely debated (Ostwald, 1894). To explain the specific
action of glycolyzing (i.e., sugar-cleaving) enzymes, Emil
Fischer (1894) proposed his Lock-and-Key Hypothesis
asserting that enzymes are rigid templates, into which
substrates must insert with the same high precision as
a key fitting into its corresponding lock. However, it was
another German chemist Eduard Büchner, who in 1897
proved that metabolism can take place outside intact
living cells. He innovated the procedure of grinding yeast
in abrasive sand, followed by passage through a paper
filter to obtain a cell-free extract. Noting the release of
CO2 bubbles after adding the resulting extract to a sucrose
solution, Büchner correctly inferred that the extract itself
acted as a catalyst, even in the absence of intact cells and
therefore any possibility of a vital force. The clean-cut
result earned Büchner the Nobel Prize in Chemistry, and
the simplicity of his protocol ushered in the modern era of
systematic biochemical research. In 1898, Duclaux suggested that the suffix ‘‘-ase’’ be used in biochemical
nomenclature to distinguish enzymes from biological
substances devoid of catalytic activity.
In his 1894 paper, Fischer asserted that among the
agents that serve the living cell, the proteins are the most
important, but the mounting evidence that enzymes were
proteins was stubbornly resisted by Richard Willstätter.
Having earned the Nobel Prize for working out the
structures of chlorophyll and heme, Willstätter held that
low-molecular-weight substances associated with proteins
were the true catalytic entities. His view persisted until the
American scientist James B. Sumner (1926) crystallized
urease, demonstrating that its catalytic power rested in the
protein itself. Subsequent work by John H. Northrop
demonstrated that proteases could likewise be crystallized
and that the protein was the sole component responsible
for catalysis. The weight of their combined findings
persuasively overwhelmed all doubters, and so doing
earned them the Nobel Prize.
13
1.2.2 Enzyme Technology
In many respects the forerunner of modern biotechnology,
the field of enzyme technology was born in Copenhagen in
1874 with the establishment of the Christian Hansen’s
Laboratory. Although mainly focusing on the production of
wax as a coating for cheeses, Hansen’s Laboratory became
the first company to market a standardized preparation of
the enzyme rennet for use in cheese-making (Tauber, 1943).
By controlling the rate and extent of milk curdling,
Hansen’s efforts greatly increased the quantity, quality, and
shelf life of European cheeses. While living in the United
States in the early 1890s, the Japanese scientist Jokichi
Takamine developed a water–alcohol extraction method to
isolate the powerful starch digesting enzyme Takadiastase.
The latter was the trade name derived by combining ‘‘Taka’’
from his name with ‘‘diastase,’’ the latter by then an already
well-known amylase preparation from the fungus
Aspergillus oryzae. His patent, granted as No. 525,823 by
the U.S. Patent Office on September 11, 1894, was the first
to teach proprietary aspects of enzyme technology.
Takamine’s efforts inspired what is now a century-old
Japanese tradition of using enzymes and highly controlled
fermentation to improve production of sugars, cheese,
beer, vinegar, bread, fermented soy products, etc., to
produce fine chemicals like monosodium glutamate, inosinic acid, and vitamins, and to isolate new drugs and antimetabolites.
It is important to recognize that fermentation science and
enzymology have profoundly altered the course of history.
A notable example is acetone-butanol fermentation. Pasteur
was the first to identify butyric acid as a fermentation
metabolite, and acetone formation was later demonstrated
by Schardinger (1905). In 1911, Fernbach and Weizmann
first reported on bacteria that produced amyl alcohol,
ethanol, and acetone as stable metabolic end-products of
potato starch fermentation. A year later, Weizmann isolated
an organism that fermented all known starches and
produced acetone in much higher yield. Those were
desperate times, and sensing the significance of his
discovery in low-residue lacquers to waterproof cloth-sided
airplanes as well as for explosives, the ardent Manchester
Zionist wrangled a promise (now known as the Balfour
Declaration) that England would support his life-long goal
of returning Jews to Palestine. British reluctance to fulfill
that promise led to the post World War II struggle that
ultimately established Israel, with Weizmann elected its first
president. Ironically, the Axis Powers relied on the immense
intellect of none other than Emil Fischer to manage the
German chemical industry during World War I. Failure of
the Axis, loss of his two sons in that great war, and
advancing cancer overwhelmed Fischer, who committed
suicide in 1919.
Today, beyond the use of enzymes in biomedicine,
enzyme technology (Tables 1.2 and 1.3) has expanded to
Enzyme Kinetics
14
TABLE 1.2 Some Commercial Applications of Enzyme Technologya
Product
Enzyme application
Animal Feed
Phytases hydrolyze abundant phytate (myo-inosital hexaphosphate) stores in plants used as
animal feed, thereby increasing the nutritional value of the feed by releasing phosphate and
bound metals from the phytate.
Rennet cleaves k-casein between Phe-105 and Met-106, thereby destroying the latter’s
ability to stabilize milk as a colloidal suspension, resulting in its calcium ion-induced
coagulation into curd and liquid whey. (Treatment of soft cheeses with hen egg white
lysozyme destroys Listeria monocytogenes, an infectious bacterial pathogen in those with
compromised cell-mediated immunity.)
Combined action of glucose oxidase and catalase removes glucose from egg whites prior to
drying into dried egg white. Glucoamylase releases b-D-glucose from 1,4-a-, 1,6-a- and 1,3a-linked glucans to yield high-glucose syrup. b-Amylases liberate maltose from barley starch
in the production of high-maltose syrup. Invertase action on sucrose yields glucose and
fructose, providing a sweeter syrup that is less apt to granulate than pure sucrose syrups.
In this three-step process, Bacillus species a-amylase acts on cornstarch to produce shorterchain polysaccharides, Aspergillus glucoamylase yields glucose, and glucoisomerase action
increases fructose content to ~42%. Because HFCS is substantially sweeter than glucose or
sucrose, less is required as a sweetener primarily in baked goods, candy, and soft drinks.
FermgenÔ protease is a proprietary fungal enzyme (pH optimum ¼ 3.0–4.5) that promises
higher rates and yields of ethanol from fermentation for corn-, milo-, or wheat-based
substrates by: (a) increasing availability of essential yeast nutrients in the form of amino acids,
peptides and free amino nitrogen; and (b) hydrolyzing protein matrices within kernels,
thereby facilitating use of otherwise hydrolysis-recalcitrant starches.
Papaya juice (rich in papain), pineapple juice (rich in bromelin), and orange juice (rich in
ficin) are all highly effective tenderizers. (Processed papaya latex extract is sold under the
brand name AccentÔ.)
Combined action of Aspergillus pectinase and Monilia diastastase greatly reduces
cloudiness, especially important for sparkling wines. In the absence of colloidal pectin,
improved filtration/pressing also increases volume by 15–20%.
Laccases (polyphenol oxidases) are used in the textile industry for dye bleaching in the
production of ‘‘stone-washed’’ denim. Cellulases are sold to the textile industry for cotton
softening and denim finishing. Alkaline pectinase, poly(vinyl alcohol)-degrading enzyme,
cutinase and catalase are also used for cotton preparation. Pectinase and hemicellulases are
used to soak and loosen bast (long and strong central) fibers for high-quality fabrics. Proteases
remove contaminating proteins from silk fibers without effect on fibroin. Transglutaminase is
used to introduce cross-links into wool, thereby strenthening fibroin strands. Amylases
remove insoluble starchs and sizing from silk and cotton to improve quality of dyeing and
printing processes.
Catalase reduces nicotine content. Glucosidases form the desired brown pigment by
hydrolysis of quercitin-rhamnoglucoside (rutin). Amylases and invertases increase glucose
and fructose content for improved taste.
Proteases (pepsin and trypsin as well as extracts of Aspergillus oryzae cultivated on rice,
elastin, and keratin) remove flesh, blood and hair from fresh hides without affecting leather’s
collagen network. Lipases remove oils that retard tanning and dyeing.
b-Xylanases are used in the treatment of paper pulp to reduce the use of chlorine for
bleaching.
Proteases (mainly subtilysin) remove proteins from food, skin, and saliva that accumulate on
clothing. Haloperoxidases are now employed to generate ‘‘color safe’’ bleaches. These
enzymes are often stabilized by intramolecular –S–S– linkages. More than half of all
detergents now contain enzymes as a proprietary constituent.
Lipases release enzymes from microbes to greatly accelerate the degradation of raw
sewerage.
Residing deep within the fissures in the surfaces of stainless steel surgical devices, prions
causing variant Creuzfeldt-Jakob Disease (vCJD) can resist standard sterilization procedures.
PrionzymeÔ (a proprietary enzyme), the Bacillus-derived MSK103 protease, as well as
a combination of proteinase K and pronase (the latter in the presence of SDS) can hydrolyze
vCJD prions. These enzymes may therefore facilitate the sterilization of neurological and
dental surgery instruments.
Cheese-making
Baking Industry
High-fructose Corn Syrup (HFCS)
Ethanol Production
Meat Tenderizing
Fruit Juices, Wine, and Beer
Textiles
Tobacco
Leather
Paper Production
Detergents
Sewage Treatment
Reducing Spread of Prion Diseases
a
The interested reader should consult Tauber (1943) for detailed early accounts of the commercial utility of enzymes. Chaplin and Bucke (1990) present
lucid descriptions of these and other more contemporary applications of enzymes in commerce.
Chapter j 1 An Introduction to Enzyme Science
15
TABLE 1.3 Several Commercially Important Enzymesa
Type
Enzymes
Carbohydrases
a-Amylases; Alkaline a-Amylase; b-Amylase; Cellulase; Cyclodextrin glycosyl tranferase; Dextranase;
a-Galactosidase; Glucoamylase; a-Glucosidase; Hemicellulase; Invertase; Lactase; Lysozyme; Naringanase;
Pectinase; Pentosanase; Pullulanase; and Xylanase.
Acid protease (Pepsin); Alkaline protease; Bromelain; Chymosin; Ficin; Neutral proteases (Trypsin, Chymotrypsin);
Papain; Peptidases; Rennet; Rennin; Subtilisin; and Thermolysin.
Triglyceridases and Phospholipases.
Amidases; Aminoacylase; Apyrase; Chlorophyllase; DNA restriction endonucleases (300þ enzymes); Feruloyl
esterases; Glutaminase; Penicillin acylase; Phytase; Phosphatases; Pregastric esterases; and Ribonucleases.
Amino acid oxidase; Catalase; Chloroperoxidase; Glucose oxidase; Glutathione peroxidase; Hydroxysteroid
dehydrogenase; Laccase; Lactate dehydrogenase; Lipoxygenase; Lysyl hydroxylase; Lysyl oxidase; Peroxidase;
Polyphenol oxidase; Sorbitol oxidase; Sulfhydryl Oxidase; and Xanthine oxidase.
Acetolactate decarboxylase; Aspartic b-decarboxylase.
RNA-dependent DNA polymerase (reverse transcriptase); Taq DNA polymerase; Vent DNA polymerase.
Fumarase; Histidase.
Glucose isomerase; Xylose (Glucose) isomerase.
Proteases
Lipases
Other hydrolases
Oxidoreductases
Decarboxylases
Polymerases
Lyases
Isomerases
a
The interested reader should consult Tauber (1943) for detailed accounts of the commercial utility of enzymes. Chaplin and Bucke (1990) present lucid
descriptions of these and other more contemporary applications of enzymes in commerce.
include the use of enzymes in the production of foodstuffs,
including hydrolysis of starch, production of glucose- and
maltose-rich syrups as well as high fructose corn starch,
derivation of glucose from cellulose, use of lactases in the
dairy industry, extended applications of enzymes in the
preparation and storage of fruit juices, and improvement of
wines, beers and distilled spirits and (Chaplin and Bucke,
1990). Enzyme technology has likewise improved production of detergents, color-safe bleach, leather and wool.
Modern biotechnology grew out of genetic engineering
in the early 1970s by the discovery of restriction enzymes
by Daniel Nathans, Hamilton Smith and Werner Arber and
the advent of recombinant DNA techniques, pioneered
largely by Paul Berg, Herbert Boyer, and Stanley Cohen.
Nothing written here can adequately encapsulate the
momentous growth of biomedicine arising from recombinant DNA. In vitro protein synthesis promises to revolutionize the production of pyrogen-free proteins, enzymes,
and antibodies for use in highly specific and low-toxicity
therapies.
For many years, enzymes found limited application in
the organic chemistry laboratory. The notable exception was
the use of pig kidney acylase for the resolution of secondary
alcohols via stereoselective ester synthesis, followed by
chromatography to separate the product.
OH
OAc
Esterase
CN
CN
H3C
H3C
Enzyme-mediated enantiomeric enrichment is discussed
in greater detail in Section 5.10. The widespread utility of
enzymes in organic and pharmaceutical chemistry has now
burgeoned over the years. Those interested in such applications should consult Biocatalysts and Enzyme Technology
(Buchholz, Kasche and Bornscheuer, 2005). Another valuable resource is Enzyme Catalysis in Organic Synthesis: A
Comprehensive Handbook (Drauz and Waldmann, 2002),
which provides tried and true methods for using enzymes in
organic synthesis, a exhaustive table of all the commercially
available enzymes, as well as comprehensive registers for
targeted searching according to enzyme, compound, or
reaction type.
1.3 DEVELOPMENT OF ENZYME KINETICS
The idea that an enzyme first combines with its substrate
was suggested by Wurtz (1880), who found that papain
appeared to form an insoluble compound with fibrin prior to
hydrolysis of the latter. O’Sullivan and Tompson (1890)
reached a similar conclusion, based on their observation that
invertase is protected by its substrate sucrose against
thermal denaturation. The theoretical basis of enzyme
kinetics was consolidated through the work of Adrian
Brown (1892, 1902) and Victor Henri (1903), whose work
on enzyme-substrate complex formation foreshadowed
(‘‘adumbrated’’, as J. B. S. Haldane (1930) put it) the
monumental paper by Leonor Michaelis and Maude Menten
(1913). Their famous relationship (Eqn. 1.2) explains the
kinetic behavior of literally thousands of enzyme-catalyzed
reactions.
v ¼
Vm
K
1þ
½S
1.2
The Michaelis-Menten treatment is based on the rapidequilibrium assumption that the concentrations of free
16
enzyme EF, free substrate SF, and enzyme-bound substrate
E$X are defined thermodynamically: Kd ¼ [EF][SF]/[E$X].
John B. S. Haldane later introduced the concept of a steadystate flux (e.g., d[E$X]/dt z 0) to enzyme kinetics and
metabolism (Briggs and Haldane, 1925; Haldane, 1930).
Both approaches sample rate behavior over the course of
many catalytic reaction cycles.8 Haldane’s use of the
steady-state approximation pre-dated the development of
non-equilibrium thermodynamic theory that now helps us to
comprehend the robust stability of steady states.
By the mid-nineteenth century, chemists Michael
Faraday and Antoine Lavoisier showed that all redox
reactions (Overall Reaction: Aox þ Bred # Ared þ Box) can
be treated as the sum of two half-reactions (Reduction HalfReaction: Aox þ e # Ared; and Oxidation Half-Reaction: Bred
# Box þ e, where e represents an electron). This concept led
to the idea that other chemical processes may likewise be
dissected kinetically into component (or elementary)
reactions.
In 1910, the German electrochemist and Nobel Laureate
Walther Nernst extended Maxwell’s theory of gases by
suggesting that fast elementary steps in solution-phase
reactions might be gainfully explored by chemical relaxation techniques. However, instrumentation of suitable
stability and sufficient sensitivity was unavailable at that
time. Recognizing a need to probe the kinetics of hemoglobin oxygenation in much greater detail, Hamilton Hartridge and Francis Roughton (1923, 1926) introduced the
rapid-mixing technique, known as continuous-flow, that
necessitated the use of 0.1–0.5 liter volumes of reactants.
Britton Chance (1943) perfected their designs through his
ingenious design of a low-volume, stopped-flow rapidmixing device that used a spectrophotometer to detect and
analyze intermediates formed transiently by horse radish
8
Initial-rate enzyme experiments analyze multiple-turnover processes
averaged over numerous catalytic turnovers. Multiple-turnover kinetic
phenomena are usually examined at low concentrations of enzyme, and
the accumulation or depletion of an enzyme-bound reactant species
during the steady-state phase is assumed to be time invariant (i.e.,
D[EX]/dt z 0). The number of turnovers occurring during an initial-rate
measurement equals D[P]/[EX] ¼ D[P]/{[P]t¼t [P]t¼0}, where [P] is
the concentration of product formed, and [EX] is the concentration of
enzyme-bound reactant over the period of measurement. The term
single-turnover process refers to events occurring over one turnover or
cycle of catalysis. As discussed in Chapter 10, single-turnover properties
are usually measured at high concentrations of enzyme using rapid
reaction techniques, such that the accumulation or depletion of an
enzyme-bound reactant species, say EX, may be detected and quantified.
Because the observed rate is a population average for many molecules
undergoing a single-turnover, the rate constants obtained are likewise
average values. The term single-molecule kinetic process refers to events
occurring at the level of individual enzyme molecules undergoing one or
more catalytic reaction cycles, observed by a suitable high-sensitivity
microscopical technique. As discussed in Chapter 12, one can also study
reactions at the single-molecule by measuring local accumulation of
product molecules generated by spatially isolated enzyme molecules.
Enzyme Kinetics
peroxidase (Reaction: Leuko-malachite Green (colorless) þ
H2O2 # Malachite Green (lmax ¼ 612 nm) þ 2 H2O).
Chance also introduced the use of analogue (and later pioneered digital) computers for modeling the kinetic behavior
of individual enzymes as well as those forming a metabolic
pathway. Exactly how the Nobel Institute has failed to
recognize Chance’s enormous contributions to modern
chemistry is an enigma.
After the discovery of the phenomenon of nuclear
magnetic resonance in 1946 by Bloch and Purcell, biological NMR spectroscopy was ushered in by Mildred Cohn
and others over the ensuing decades. Likewise, surging
interest in sonar and shock-wave technology during World
War II, coupled with the theory of pressure-induced
chemical relaxation (Einstein, 1920) provided the impetus
for the investigation of individual steps (elementary reactions) within multi-step kinetic mechanisms. Fast reaction
methods, especially those pioneered by Nobel Laureates
Manfred Eigen (temperature-jump technique), Ronald
G. W. Norrish (shock-tube and pressure-jump techniques)
and George Porter (flash photolysis), completely revolutionized experimental chemical kinetics.
Although somewhat beyond the current discussion, one
cannot minimize the impact of developments in physical
organic chemistry on the emergence of enzyme science. The
British chemist Keith Ingold introduced the terms electrophile for an electron-seeking functional group, nucleophile
for nucleus-seeking functional group, tautomerism for ketoenol isomeric rearrangements, and inductive effect to
account for electronic effects of nearby entities on functional group reactivity. A fundamental advance was his
conceptualization of the respective dissociative and associative features of SN1 and SN2 nucleophilic substitution
mechanisms at saturated carbon bonds. (Later work disclosed that corresponding SN1 and SN2 mechanisms are also
at play in phosphotransfer reactions.) Another Briton,
Ronald Bell, connected the acid base theory of his mentor
Brønsted to the origins of hydrogen isotope effects and
correctly predicted that the kinetic isotope effect should be
maximal when the proton is half-transferred in the reaction’s transition state. Perhaps the most influential of Bell’s
contributions was his development and understanding of
quantum mechanical tunneling, or as he called it the tunnel
correction for isotope effects involving proton (and hydride)
transfer processes. With their later keen interest in understanding biological proton transfer, Bell’s disciples John
Albery and Jeremy Knowles found warm acceptance of
their novel ideas on enzyme catalysis.
Over the past half-century, enzyme kinetics has matured
into a highly sophisticated and innovative discipline.
Although the current state of any field is the sum of contributions of countless investigators, the following scientists
made advances so notable that they personify the field:
Robert Abeles – Mechanism-based inhibitor design;
Cobalamin-dependent reactions; Robert Alberty – pH
Chapter j 1 An Introduction to Enzyme Science
17
TABLE 1.4 Nobel Prizes Awarded for Research in Enzyme Sciencea
Year
Laureate
Award
Cited achievement
2009
2009
2009
2006
2004
2004
2004
2000
1997
1997
1997
1994
1994
1993
1992
1992
1989
1988
1988
1988
1988
1982
1982
1978
1978
1978
1978
1975
1972
1972
1972
1971
1970
1970
1964
1964
1961
1959
1959
1955
1953
1953
1947
1947
1947
1946
1946
1937
1931
1929
1929
1922
1907
Venkatraman Ramakrishnan
Thomas A. Steitz
Ada E. Yonath
Roger Kornberg
Aaron Ciechanover
Avram Hershko
Irwin Rose
Paul Greengard
Paul Boyer
John Walker
Jens Skou
Alfred Gilman
Martin Rodbell
Kary Mullis
Edmond Fischer
Edwin Krebs
Sidney Altman
Thomas Cech
Johann Deisenhofer
Robert Huber
Hartmut Michel
Sune Bergström
Bengt Samuelsson
Peter Mitchell
Werner Arber
Daniel Nathans
Hamilton Smith
John Cornforth
Christian Anfinsen
Stanford Moore
William Stein
Earl Sutherland
Louis Leloir
Julius Axelrod
Konrad Bloch
Feodor Lynen
Melvin Calvin
Arthur Kornberg
Severo Ochoa
Hugo Theorell
Hans Krebs
Fritz Lipmann
Carl Cori
Gerty Cori
George Wald
James Sumner
John Northrop
Albert Szent-Györgyi
Otto Warburg
Arthur Harden
Hans von Euler-Chelpin
Otto Meyerhof
Eduard Buchner
Chemistry
Chemistry
Chemistry
Chemistry
Chemistry
Chemistry
Chemistry
Med/Phys
Chemistry
Chemistry
Chemistry
Med/Phys
Med/Phys
Chemistry
Med/Phys
Med/Phys
Chemistry
Chemistry
Chemistry
Chemistry
Chemistry
Med/Phys
Med/Phys
Chemistry
Med/Phys
Med/Phys
Med/Phys
Med/Phys
Chemistry
Chemistry
Chemistry
Med/Phys
Chemistry
Med/Phys
Med/Phys
Med/Phys
Chemistry
Med/Phys
Med/Phys
Med/Phys
Med/Phys
Med/Phys
Med/Phys
Med/Phys
Med/Phys
Chemistry
Chemistry
Med/Phys
Med/Phys
Chemistry
Chemistry
Med/Phys
Chemistry
Ribosome structure and mechanism
Ribosome structure and mechanism
Ribosome structure and mechanism
Mechanism of transcription (RNA polymerase)
Mechanism of enzymatic ubiquitination
Mechanism of enzymatic ubiquitination
Mechanism of enzymatic ubiquitination
Signal transduction and brain protein kinases
ATP synthase rotary catalysis mechanism
ATP synthase structure
Discovery of sodium, potassium ATPase
Signal-transducing GTP-regulatory enzymes
Signal-transducing GTP-regulatory enzymes
Polymerase chain reaction
Protein kinases
Protein kinases
Catalytic RNA
Catalytic RNA
Structure of a photosynthetic reaction center
Structure of a photosynthetic reaction center
Structure of a photosynthetic reaction center
Prostaglandin biosynthesis
Prostaglandin biosynthesis
Chemiosmotic principle
Discovery of restriction enzymes
Discovery of restriction enzymes
Discovery of restriction enzymes
Stereochemistry of enzyme-catalyzed reaction
RNase folding
RNase sequence and active-site chemistry
RNase sequence and active-site chemistry
Discovery of 39,5-cyclic-AMP
Structure and biosynthesis of sugar nucleotides
Enzymatic synthesis of epinephrine
Cholesterol metabolism
Fatty acid metabolism
Photosynthesis
Enzymatic synthesis of DNA
Enzymatic synthesis of RNA
Mechanisms of redox enzymes
Citric acid pathway
Coenzyme A and fatty acid enzymology
Enzymatic synthesis of glycogen
Enzymatic synthesis of glycogen
Retinal cis-trans isomerization in visual processes
Urease crystallization
Protease crystallization
Vitamin C and catalysis of fumaric acid
Mode of action of respiratory enzymes
Sugar fermentation pathway
Fermentative enzymes
O2 and lactic acid metabolism
Cell-free enzyme-catalyzed reactions
a
Although receptor-mediated endocytosis and prions have little to do with enzymes, their respective discoverers, Michael Brown (Nobel Laureate in
Medicine and Physiology, 1987) and Stanley Prusiner (Nobel Laureate in Medicine and Physiology, 1997), both received their post-doctoral research training
in enzymology under the late Earl R. Stadtman.
18
kinetics; Bisubstrate enzyme kinetics; Thermodynamics of
ATP hydrolysis of biochemical reactions; Application of
Legendre transforms in biochemical thermodynamics;
Christian Anfinsen, Stanford Moore and William Stein –
Ribonuclease structure and folding, identification of catalytic residues; John Albery and Jeremy Knowles – Novel
isotopic approaches for defining the energetics of the triosephosphate isomerase and proline racemase reactions;
Enzyme evolution, Catalytic efficiency, and Catalytic
perfection; Max Bergmann and Joseph Fruton – Poly-site
binding theory of enzyme specificity; Introduction of
synthetic N-carbobenzoxy-peptides as alternative substrates
for proteases and peptidases; Paul Boyer – Multi-substrate
enzyme kinetics; Definition of kinetic reaction mechanisms
through the novel application of isotope exchange
measurements at thermodynamic equilibrium; Oxygen-18
tracer methods in carboxyl- and phosphoryl-group transfer
reactions; ‘‘Binding-Change Mechanism’’ for rotary catalysis of ATP synthase (see Table 1.4: Nobel Laureates);
Britton Chance – Invention of the stopped-flow technique;
First spectral detection of enzyme reaction intermediates;
First application of computers to simulate enzyme reaction
kinetics; Development of the Theorell-Chance bisubstrate
kinetic mechanism; W. Wallace Cleland – Systematic
enzyme nomenclature of multi-substrate enzyme kinetics;
Steady-state treatment of isotope exchange kinetics;
Development of exchange-inert metal-nucleotide complexes; Development of equilibrium perturbation technique
to evaluate kinetic isotope effects for detecting rate-limiting
chemical steps; Mildred Cohn and Albert Mildvan –
Oxygen-18 probes of P–O and C–O bond cleavage in
phosphotransfer reactions; Development of NMR-based
distance measurements using proton relaxation in paramagnetic environments; NMR approaches for defining
enzyme exchange kinetics; Keith Dalziel – Development of
the F-parameter method for discriminating the order of
substrate binding by bisubstrate enzymes; Edward Dennis,
Pierre Desnuelle, Michael Gelb, Mahendra Jain and Robert
Verger – Use of nonionic detergents and Langmuir troughs
to investigate interfacial catalysis by lipases and phospholipases; Lipase processivity; Zacharias Dische – Discovery
of allosteric feedback inhibition; Pierre Douzou and
Anthony Fink – Development of ultra-low temperature
(cryoenzymology) techniques to investigate enzyme kinetic
properties; Manfred Eigen – Chemical relaxation process;
Temperature-jump technique; Prion protein polymerization
(see Table 1.4: Nobel Laureates); Fritz Eckstein, Jeremy
Knowles, David Usher and Martin Webb – Stereochemical
probes of phosphomonoester- and phosphodiester-utilizing
reactions; Alan Fersht – Site-directed mutagenesis as
mechanistic probes; Mechanisms for kinetic proofreading
by aminoacyl-tRNA synthetases; Novel approaches for
defining protein folding mechanisms; Carl Frieden – pH
kinetics of fumarase reaction; Three-substrate enzyme
kinetics; Kinetic aspects of enzyme cooperativity and
Enzyme Kinetics
hysteresis; Development of KINSIM and FITSIM software
for simulating enzyme rate processes; Herbert Fromm – Use
of reversible inhibitors (including product inhibitors,
alternative substrate inhibitors, as well as competitive
inhibitors) to distinguish multi-substrate kinetic mechanisms;
Implications of abortive complex formation in enzyme
kinetics; Definition of kinetic reaction mechanisms (with
Boyer) through isotope exchange measurements at thermodynamic equilibrium; Constant-ratio approaches for
analyzing three-substrate enzyme kinetics; Fallacy of adenylate energy charge hypothesis for ATP-utilizing/regenerating enzymes; Quentin Gibson – Development of stoppedflow rapid mixing instrumentation; Heme-protein kinetics;
Herbert Gutfreund – Fast reaction kinetics of enzyme
reactions; Kinetic criteria (with P. Boon Chock) for evaluating substrate channeling; Gordon Hammes – Temperaturejump reaction techniques to enzyme systems; Fast reaction
kinetics of complex multi-enzyme processes; Brian
Hartley – Chymotrypsin catalysis; Enzyme burst method for
detecting enzyme-bound, covalent reaction intermediates;
Charles Huang – Multisubstrate enzyme kinetics; Models
for calcium ion complexation in calmodulin mediated activation of target enzymes; Kinetic analysis of allosteric
enzymes; William Jencks – Catalytic strategies in chemistry
and enzymology; Conceptual basis for catalytic antibodies,
Energetics and mechanism of calcium ion pump; Kaspar
Kirschner – Fast reaction kinetics of allosteric enzymes;
Daniel Koshland – Induced-fit hypothesis; Sequential model
for cooperativity of allosteric enzymes; Role of orbital
alignment (Orbital Steering) in enzyme catalysis; Keith
Laidler – Application of absolute rate theory to enzyme
systems; Temperature and immobilization effects on
enzyme kinetics; Richard Lerner and Peter Schultz –
Development of catalytic antibodies, based on a prediction
by W. P. Jencks; Vincent Massey – pH Kinetics of fumarase;
Kinetic and mechanistic approaches in flavoenzyme catalysis; Peter Mitchell – Chemiosmotic principle of transmembrane gradients (see Table 1.4: Nobel Laureates);
Jacques Monod, Pierre Changeaux and Jeffries Wyman –
Concerted transition model for allosteric interactions and
cooperativity; Dexter Northrop – Two-site ping-pong
kinetics; Exploiting the Swain-Schaad relationship to isolate
and evaluate intrinsic kinetic isotope effects; Dieter Palm,
Bryce Plapp and Judith Klinman – Kinetic isotope effects in
enzyme-catalyzed hydride transfer; Role of quantum
mechanical tunneling in hydride transfer; Arthur Pardee and
Edwin Umbarger – Kinetics and feedback inhibition of
allosteric enzymes; Ephraim Racker – First demonstration
that covalent enzyme-substrate compounds are formed
during enzyme catalysis; Michael Raftery – Early application of secondary kinetic isotope effects to detect the oxacarbenium ion intermediate formed in lysozyme catalysis;
Irwin Rose – Isotopic probes of enol intermediates in
isomerases; Isotope trapping methods; Dynamic stereochemical probes (or positional isotope exchange);
Chapter j 1 An Introduction to Enzyme Science
Elucidation of the ubiquitin ligase mechanism (see
Table 1.4: Nobel Laureates); Vern Schramm – Application of
multiple kinetic isotope effect data to the rationale design of
transition-state analogues for uses as specific, high-affinity
enzyme-targeted drugs; Earl Stadtman – Kinetic and regulatory behavior of signal transduction cascades via post
translational modification, as demonstrated in his pioneering
studies of enzyme-catalyzed adenylylation/deadenylylation
of Escherichia coli glutamine synthetase; Edwin Taylor,
David Trentham, Clive Bagshaw and Martin Webb –
Mechanoenzyme kinetics of actomyosin, as probed by fast
reaction kinetics, ‘‘photo-caged’’ ATP, and continuous
assay with fluorescent phosphate-binding protein; Hugo
Theorell – Bisubstrate reaction kinetics of redox enzymes
(see Table 1.4: Nobel Laureates); Frank Westheimer –
Stereochemistry NADH hydride transfer; Stereochemistry
of phosphoryl transfer, including pseudorotation; Kinetic
isotope effects; Photoaffinity labeling of enzyme active
sites; Bioinorganic reaction mechanisms; Richard Wolfenden – Development of a rational basis for analyzing
transition-state inhibitor potency; Catalytic proficiency; and
Jeffrey Wong – Theoretical treatment of steady-state enzyme
kinetics; Alternative substrate kinetics.
Finally, those familiar with enzyme kinetics know that
the complexity of certain enzymes generated such
compelling interest that some enzyme chemists made
career-long commitments to the study of a single enzyme or
pathway. So strong was their attachment to their favorite
enzyme that the late Ephraim Racker once told the author
that he was convinced that the perceived importance of an
enzyme was often a manifestation of the interesting
personalities investigating that enzyme. He was particularly
fond of the humanistic saying that ‘‘Interesting people make
for interesting enzymes.’’ In this respect, the above list is
admittedly incomplete and fails to acknowledge the
immense contributions of so many other creative and
interesting scientists.
1.4 THE CONCEPT OF A REACTION
MECHANISM
The chief ambition of enzyme chemists is to obtain the most
complete description possible of an enzyme-catalyzed
reaction. An enzyme’s overall catalytic mechanism may be
subdivided into four parts:
1. Chemical Mechanism – A reaction scheme showing all
bond-breaking/-making steps, rearrangements, transition state(s), as well as the stereochemistry of partial
and overall reactions.
2. Kinetic Mechanism – A scheme accounting for the
time-dependent accumulation and breakdown of each
enzyme-bound species, including the energetics of any
rate-determining step(s).
19
3. Structural Mechanism – An atomic-level model
showing the structural basis for catalytic facilitation of
the chemistry of substrate-to-product interconversion
as well as the physics of substrate adsorption and
product release.
4. Regulatory Mechanism – A scheme offering a detailed
understanding of activator and inhibitor effects that are
a direct consequence of binding cooperativity, allosteric
interactions with activators and/or inhibitors, post-translational modification, etc.
Undertaking such investigations begins with elucidation of
a chemical reaction mechanism explaining all of the bondbreaking and bond-making steps needed to transform
substrate(s) into product(s), as well as all detectable
elementary reactions comprising the kinetic scheme of
enzyme interactions with substrates, intermediates, and
products. Although many studies are initiated with the
convenient use of unnatural substrates that are chromogenic or fluorogenic (i.e., the products of these weakly
absorbing or fluorescing substrates have quantifiable
absorbance or fluorescence spectra), these studies should
ideally be carried out with the natural substrates to fully
understand the biological role of the enzyme under investigation. (In fact, altered reactivity of alternative substrates
must always be anticipated.) The chemical and kinetic
mechanisms must be consistent with the reaction’s overall
stoichiometry, its stereochemistry, its kinetic and thermodynamic properties, the location and energetics of rate
determining step(s), the structures of detected intermediates as well as any inferred transition state(s), as well as
effects of temperature, pH, ionic strength, and solvent. The
structural mechanism begins with high-resolution structures of the free enzyme as well as it complexes with
reaction substrate(s) and product(s), as well as any activators or inhibitors of interest. But a structural interpretation is incomplete unless it unifies the chemical, kinetic,
and regulatory mechanisms. The regulatory mechanism
should explain how an effector molecule lowers (activation) or raises (inhibition) the activation energy of one or
more steps in the catalytic reaction cycle. Likewise, the
effect of any post-translational modifications should be
reconciled with changes in the catalytic reaction
mechanism.
The optimal approach for integrating such information
is to construct rival hypotheses that make testable
predictions connecting structure, energetics, and kinetics.
Ideally, these rival explanations will result in kinetically
distinguishable properties. Enzyme chemists make
strenuous demands on structural and chemical information, and kinetic data often offer additional constraints for
deciding on the most likely of rival reaction mechanisms.
Modern enzymology has benefited enormously from the
atomic-level molecular structures, as provided by X-ray
crystallography and high-resolution, multidimensional
20
NMR spectroscopy. Even so, while structural biologists
have glimpsed various stages of catalysis, there is no such
thing as a tell-all ‘‘motion picture’’ of even the simplest
catalytic process. These days, there are those enzyme
chemists who won’t believe anything without first seeing
it, while others don’t see anything without first believing
it. While we would desire to view catalysis from
a vantage point of quantum mechanics, the chief obstacle
to applying quantum mechanical approaches is that
enzymes are complex structures, frequently possessing
10,000–15,000 atoms, the positions of which are rarely
known with adequate accuracy. Enzyme structures are
also strongly influenced by seemingly countless noncovalent bonding interactions, and each non-covalent
interaction contributes a relatively small increment to the
conformational energy associated with an enzyme’s
catalytically active conformation. Dealing with so many
weak interactions remains a daunting challenge for
computer software developed to treat far simpler molecules. Even when quantum mechanical calculations are
limited to a small segment or region within an enzyme
(say the active site region), quantum mechanical and
molecular mechanical models can quickly become
unwieldy. Even so, one can safely predict that, with
advances in computer-based calculations, quantum
mechanics may eventually prevail, as it promises to offer
the ultimate picture of catalysis.
An enzyme mechanism must provide much more than
just the changes in covalent structure. A mechanism must
also explain the enzyme’s actions during catalysis – all
substrate binding interactions, all stereochemical transformations, all pathways for product release, solvation
changes in active site, etc. The same also goes for changes
in coenzymes, cofactors and metal centers. Enzyme kineticists also seek to understand those structural, dynamic, and
catalytic changes that are the basis of an enzyme’s regulatory behavior. Allosteric activators and inhibitors of
enzymes have the effect of respectively lowering and raising
reaction barrier(s), as do the activating and inhibitory effects
of post-translational covalent modifications of enzymes.
Allosteric transitions often involve a manifold of protein
conformational states, the complexity of which imposes
such kinetic ambiguity that one cannot reach penetrating
conclusions about how an allosteric modifier alters
catalysis.
1.4.1 Chymotrypsin: The Prototypical
Biological Catalyst
Chymotrypsin was among the earliest crystallized
enzymes, and its purity and abundance stimulated great
interest in this amidohydrolase. The probable catalytic
mechanism for chymotrypsin has been worked out during
the past half-century of intensive investigation. This
Enzyme Kinetics
enzyme cleaves peptide bonds within peptides and
proteins, acting preferentially at sites where the carboxyldonating amino acid residue has a hydrophobic side-chain.
The reaction is facilitated by push–pull proton transfer
involving specific imidazole, carboxyl, and hydroxyl
groups that are common to hundreds of other mechanistically related enzymes in the ‘‘serine’’-protease superfamily. An acyl-serine intermediate permits one product
(designated by the R-group in Fig. 1.3) to dissociate, such
that water can replace the departing amino group in
a manner that leads to hydrolysis of the peptidyl acylenzyme and subsequent release of the second peptide
fragment (designated by I9). Enzyme chemists are
reasonably confident of the general outline of the steps
illustrated in Fig. 1.3, especially in the light of the wealth
of structural, chemical, and kinetic information gleaned
from persistent and systematic investigation.
Figure 1.4 illustrates the following key points about
serine-protease (and serine esterase) catalysis: (a) the
substrate and enzyme are structurally complementary with
respect to each other, with specificity determined by the
nature of charged residues deep within the active site;
(b) the mechanism exploits general base catalysis (see
Section 7.3.9: Brønsted Theory of Acid and Base Catalysis) by imidazole to activate the hydroxyl group of the
active-site serine residue; (c) the latter exhibits nucleophilic catalysis, as evidenced by the formation of a tetrahedral adduct; (d) the enzyme stabilizes the tetrahedral
transition state (and the transient covalent intermediate)
through hydrogen bonding between enzyme and intermediates, particularly within the oxy-anion hole and by the
electrostatic environment, provided in part by Asp-102;
(e) the reaction proceeds onward by means of general acid
catalysis that facilitates the departure of the leaving group
to form the acyl-enzyme (covalent) intermediate and
departure of the amine (or alcohol) leaving group; and (f)
the remaining steps in the catalytic cycle are formally the
reverse of the above steps, resulting in hydrolysis of the
acyl-enzyme, which commences with the imidazole group
activating water by general base catalysis, so as to facilitate nucleophilic attack by water at the carbonyl carbon
atom. A major limitation relates to an almost exclusive
reliance on synthetic chromogenic substrates (i.e., those
generating a change in the substrate’s or product’s UV/
visible spectrum upon peptide bond cleavage). Virtually
nothing is known about the details (e.g., steady-state and
fast kinetics, reaction cycle energetics, hydrogen bonding
of the water substrate, conformational dynamics, as well
as the formation and turnover of key intermediates)
describing chymotrypsin catalysis when proteins serve as
substrates. In this respect, the mechanism shown in
Fig. 1.4 is still somewhat incomplete.
It is also worth emphasizing that despite the many steps
in the catalytic reaction cycle, chymotrypsin is a powerful
catalyst, as evidenced by the infinitesimally low
Chapter j 1 An Introduction to Enzyme Science
21
FIGURE 1.3 Likely mechanism for chymotrypsin catalysis. Form-1 is the substrate-free enzyme, with its catalytic triad consisting of the
solvent-inaccessible, side-chain carboxyl group of Aspartate-102, the side-chain imidazole group of Histidine-57, and the side-chain hydroxyl group
of Serine-195. The location of these functional groups within the active-site cleft is depicted in the accompanying chymotrypsin structure (inset on
upper right), based on the X-ray crystallographic work of David Blow. After substrate binding to an initial, reversible enzyme-substrate Michaelis
complex, the catalytic triad in Form-2 facilitates nucleophilic attack by activating the otherwise poorly reactive serine hydroxyl group. A key point
is that partial bond formation, and the resulting hydroxyl group polarization is sufficient to accelerate catalysis; formal ionization of the serine
hydroxyl group is unlikely, because the alkoxide (pK near 15) is a far stronger base than the imidazole (pK¼6). Upon nucleophilic attack, the
carbonyl group is converted to the tetrahedral ‘‘oxy-anion’’ intermediate (Form-3), a transition-state that is stabilized by two hydrogen bonds (dashed
lines) supplied by two backbone peptide N–H groups from Glycine-193 and Serine-195. The oxy-anion spontaneously rearranges to form the covalent,
acylated enzyme (Form-4). After the amine-containing product departs, the reaction cycle then proceeds with its second phase, commencing with the
entry of water molecule into the active site (Form-5). Nucleophilic attack by this water molecule results in the second tetrahedral intermediate (Form-6),
again stabilized by the hydrogen bond network. This second oxy-anion species spontaneously rearranges to form the reversible Michaelis complex
(Form-7), with the active site occupied by the carboxyl group-containing product. The same double-displacement, or Ping Pong, pathway is likely to
apply to hundreds of other members of the ‘‘serine’’ protease superfamily, including trypsin, elastin, and thrombin. Specificity is achieved by interactions with other substrate-recognition residues not indicated here.
uncatalyzed rate (k ¼ ~1013 s1 at pH 7 at 298 K) of
peptide bond hydrolysis compared to the corresponding
reaction carried out in the presence of chymotrypsin (kcat ¼
~10 s1). The catalytic rate enhancement for chymotrypsin
is thus an astonishing 100,000,000,000,000! As discussed
throughout this textbook, the occurrence of covalent reaction intermediates during catalysis in no way impedes an
enzyme. Enzyme chemists also learned for certain that
enzymes exploit a myriad of intermediates to achieve such
high catalytic rate enhancements.
Finally, chymotrypsin is first biosynthesized as the
inactive storage form chymotrypsinogen. The latter is an
example of a zymogen – an inactive enzyme precursor, from
which an active enzyme can be generated enzyme-catalyzed
Enzyme Kinetics
22
proteolysis. While still elongating from the ribosome, the
nascent polypeptide chain is directed to and translocated into
the lumen of secretory granules, where it is oxidatively
processed to introduce essential –S–S– bonds. Upon
hormone-stimulated release into the small intestine,
chymotrypsinogen is then cleaved between residues 15 and
16 by trypsin to yield two polypeptide chains that remain
linked by means of a single disulfide bond. This peptide
cleavage process (Reaction: Chymotrypsinogen þ H2O #
p-Chymotrypsin) generates an intermediate species, known
as p-chymotrypsin, which has an imperfectly formed active
site and is hence a feeble catalyst. p-Chymotrypsin then
undergoes autocatalysis (see Section 3.8.4), with peptide
bond cleavage (Reaction: p-Chymotrypsin þ H2O #
Chymotrypsin þ Peptides) achieved through the action of
chymotrypsin and itself forms a second fully active chymotrypsin molecule. The latter consists of three disulfide-linked
polypeptides: Chain-A, the N-terminal region ending at
residues 1 to 14; Chain-B, the longest chain comprising
residues 16 to 146; and Chain-C, comprising the C-terminal
region, beginning at residue 149. Note that two short
peptides, consisting of residues 14–15 and 147–148, have no
catalytic role and are released to form the active enzyme.
Fully active chymotrypsin possesses an ‘‘oxy-anion hole’’
that accommodates the negatively charged tetrahedral
intermediates already described in Fig. 1.3, thereby affording
yet another way to promote catalysis by stabilizing an
obligatory reaction intermediate.
1.4.2 Ribozymes
From the earliest times, enzymes were always associated
with proteins, and the inspired work of Nobel Laureate
John B. Sumner on the crystallization of jack bean urease
Catalytic
RNA
Loop-3
,
N N
,
N
N
Stem-3
,
N N
A U,
X , , , ,
A
,
,
5
3
N N
, , , , A CleavageN N
Loop-1
N N N N G
Site
N N N N
Loop-2
,
N N N N
C Stem-1
Stem-2 A
U
G
N A
placed such ideas on a firm footing. Even so, Nobel
Laureates Thomas Cech and Sidney Altman demonstrated
that certain RNA molecules are highly efficient catalysts
for RNA self-splicing, phosphotransfer, and even
peptide bond formation (Altman, 1993; Cech, 1993). These
catalytic RNA molecules, also known as ribozymes,
often achieve rate enhancements approaching 1011. The
hammerhead-shaped ribozyme (Fig. 1.5) was the first RNA
motif observed to catalyze sequence-specific self-cleavage
by a magnesium ion-dependent transesterification. Containing only around 30 nucleotides in their catalytic cores,
these ribozymes are the smallest of the catalytic RNA
molecules. These enzymes display Michaelis-Menten
kinetics in their action on substrates (see Section 5.6:
Ribozyme Kinetics), with Michaelis constants (Km) values
ranging from 20 to 200 nM and turnover numbers (i.e.,
kcat) in the range of 0.03 s1. Product release is generally
fast, suggesting that the rate-determining step is phosphodiester bond-scission.
Ribozyme-mediated phosphoryl transfer appears to
involve destabilization of the substrate’s ground-state
(see also Section 1.5.4: ‘‘Reacting Group Approximation, Orientation and Orbital Steering’’ under Section
1.5: Explaining the Efficiency of Enzyme Catalysis).
Magnesium ion complexation and hydrogen bonding
stabilize the negative charge that develops on the
leaving group during entry of the nucleophile. This
transesterification reaction is mechanistically analogous
to that used in the mRNA spliceosome as well as in
other DNA topoisomerase and transposition reactions.
The true catalytic nature of the ribozyme was demonstrated by the discovery that the RNA component of
RNase P catalytically processed tRNA precursors
(Altman, 1993).
NH2
5'-End
N
O
O
P
N
O
O
O
HO
P
O
FIGURE 1.4 Generalized base-pair structure of hammerhead ribozymes. Shown are consensus nucleotide residues (marked G, C, A, U)
within the central ring consisting of 17 nucleotides (aqua) as well as variable nucleotides (N). This secondary structure is stabilized by three runs of
hydrogen-bonded nucleotide pairs forming the same type of ‘‘stem-andloop’’ structural elements that are frequently observed in folded, singlestranded messenger RNA and ribosomal RNA. The central ring and the
variable-length loops (indicated by dashed lines) facilitate folding into
a compact, sphere-shaped tertiary structure. Self-cleavage site is indicated
in red.
Me2+(OH2)4
O
O
O
O
5'-Leaving
Group
Scheme 1.4
Scheme 1.4 illustrates the likely catalytic path for selfsplicing reaction of group-II introns, which requires the
proper folding of intronic RNA into its enzymatically
Chapter j 1 An Introduction to Enzyme Science
23
Over the past century, biochemists have discovered
literally thousands of different enzyme catalyzed reactions. A compilation by Purich and Allison (2002) puts
the number at nearly 7,000 unique catalytic activities, but
data from various genome projects suggest there are
likely to be another three to five thousand more enzymes
whose reactions remain to be defined. A large number
appear to be protein kinases, receptor-linked GTP-regulatory proteins, and chromatin remodeling enzymes, as
well as enzymes mediating micro-nutrient metabolism.
Based on the ways that enzymes break, rearrange, and
form covalent bonds, and guided largely by organic
chemical principles that distinguish reaction types, the
Enzyme Commission defined the following classification
scheme.
Enzyme science has traditionally focused on the organic
chemistry of biochemical reactions, particularly the changes
in covalent bonding as substrate is transformed into product.
This rewarding enterprise helped to establish the role of
countless covalent and ionic intermediates as well as the
role of coenzymes and other cofactors. It’s a historic fact,
however, that Boyer’s discovery of the ATP synthase
mechanism was delayed by the failure of researchers to
realize that the driving force for ATP synthesis was not
a high-energy covalent intermediate, as ironically he had
himself originally proposed (Boyer, 2002). Peter Mitchell’s
chemiosmotic principle ultimately illuminated the need to
rationalize how Gibbs free energy, stored in the form of
a transmembrane proton gradient, can drive ATP synthesis
from ADP, Pi, and Hþ, and vice versa.
Contemporary biochemistry has demonstrated time and
time again that many reactions have: (a) substrate-like or
product-like protein conformational states differing only in
their non-covalent bonding interactions: or (b) substratelike or product-like state corresponding to transmembrane
solute gradients. Various ATP- and GTP-dependent molecular motors, for example, rely on the free energy of ATP
hydrolysis to drive protein conformational changes, which
in turn drive processes like muscle contraction, organelle
trafficking, and cell crawling. Structural metabolism
represents the ceaseless building-up and tearing-down of the
cell’s macromolecular and supramolecular structure
through the ATP- and GTP-dependent affinity-modulated
interactions of chaperonins and proteasomes, molecular
motors, pumps, latches, and switches. Other reactions, such
as the facilitated exchange of tightly bound protein–ligand
complexes or membrane carriers, strictly involve changes in
non-covalent bonding and proceed without the breaking/
making of even a single covalent bond. In short, mechanoenzyme catalysis involves non-covalent substrate-like
and product-like states, and the failure to include these in
describing mechanoenzyme reaction has led to confusion in
enzyme nomenclature and classification.
To provide a rational framework for the systematic
classification of enzymes, including mechanoenzymes,
Purich (2001) offered a new definition for an enzyme:
Class-1: Oxidoreductases – catalyze oxidation/reduction
reactions.
‘‘An enzyme is a biological catalyst for making and/or
breaking chemical bonds.’’
active form. The reaction mechanism probably
commences with metal ion-assisted loss of a proton ribose
29-OH, allowing the incipient 29-alkoxide to attack the
phosphodiester. Upon forming a pentavalent oxyphosphorane intermediate (with opposing nucleophile and
exiphile), the rate-limiting step is likely to involve P–O
bond scission. The active-site metal ion both facilitates
intronic RNA folding as well as stabilizes the transition
state. With the notable exception that pancreatic Ribonuclease A employs imidazole group in place of a metal
ion to polarize the 29-OH, the catalytic reaction cycles of
RNase and self-splicing reaction of group-II introns are
remarkably similar.
The discovery of catalytic RNA reminds us that one
should not dismiss the possibility that other biological
substances (e.g., polysaccharides, complex lipids, etc.) may
prove to be biological catalysts. The phenomenon of
micellar catalysis, for example, is already firmly rooted in
modern organic chemistry. In fact, some micellar catalysts
even exhibit chiral recognition (i.e., the capacity to combine
with and transform substrate molecules in a stereoselective
manner). The likely role of biomembranes in catalysis
remains to be determined.
1.4.3 Mechanoenzymes
Class-2: Transferases – catalyze group-transfer reactions.
Class-3: Hydrolases – catalyze hydrolytic cleavage of
covalent bonds.
Class-4: Lyases – catalyze addition and elimination of functional groups to unsaturated and saturated carbon atoms.
Class-5: Isomerases – catalyze rearrangement of atoms or
groups of atoms.
Class-6: Ligases – catalyze joining of molecules or functional groups.
While appearing to be no more encompassing than
existing definitions of enzyme catalysis, the crucial
difference lies in the use of chemical in place of covalent
to describe the bonding changes. This definition
acknowledges those enzymes catalyzing the interconversion of non-covalent substrate- and product-like states or
conditions:
Interaction State-1 + ATP + H20
Interaction State-2 + ADP+ Pi
Scheme 1.5
Enzyme Kinetics
24
Biological catalysis of this type is observed in instances
where the substrate is a protein with a very slowly dissociating ligand. An example is the adenine nucleotide
exchange reaction of the cytoskeletal protein actin.
Hydrolysis of actin-bound ATP during cell motility leads to
the formation of tightly bound Actin$ADP. Spontaneous
exchange of solution-phase ATP with Actin$ADP to
regenerate Actin$ATP is too slow to sustain the high filament assembly rates (400–500 monomer/filament/sec)
needed to sustain cell motility. To overcome this kinetic
obstacle, motile cells have high concentrations of profilin,
a 15-kDa regulatory protein that catalyzes the following
protein–ligand exchange reaction:
Profilin + Actin·ADP
Profilin·Actin·ADP
[Profilin·Actin·__ ]‡ + ADP
Profilin·Actin·ADP
[Profilin·Actin·__ ]‡ + ATP
Profilin·Actin·ATP
Profilin·Actin·ATP
Profilin + Actin·ATP
Scheme 1.6
Red- and blue-colored nucleotides are used in Scheme
1.6 to indicate that the reaction is one of physical exchange,
as opposed to the transfer of a phosphoryl group from
unbound ATP to form actin-bound ATP. Profilin accelerates
this reaction by a factor of 150, and profilin’s action is
without effect on the exchange reaction equilibrium. As
shown in Fig. 1.5, profilin binds preferentially to nucleotidefree actin, approximately 12-times more tightly than to
actin$ATP, and 72-times more tightly than to actin$ATP
(Selden et al., 1999). Profilin’s preferential interaction with
nucleotide-free actin explains its ability to promote
+
X+
Uncatalyzed
nucleotide exchange, and this property is indistinguishable
from the cardinal feature of all catalysts, namely transitionstate stabilization. Such considerations demonstrate unambiguously that biological catalysis can take place without
the breaking and making of covalent bonds.
In his timeless book The Nature of the Chemical Bond,
Linus Pauling (1945) offered the following definition that
has guided my thinking about enzyme catalysis:
‘‘We shall say that there is a chemical bond between two
atoms or groups of atoms in case the forces acting between
them are such as to lead to the formation of an aggregate
with sufficient stability to make it convenient for the chemist
to consider it as an independent molecular species.’’
Significantly, Pauling made no mention of covalent bonds,
stressing instead the unifying nature of chemical bonds.
That many protein conformational states and numerous
protein–ligand complexes have been shown to be sufficiently long-lived to exhibit chemically definable properties
suggests that transformations in these non-covalent interactions ought to be treated as chemical reactions. And with
modest tinkering, Pauling’s definition of a chemical bond
can be extended to include the persistent, definable position
of a solute relative to the inner and outer faces of
a membrane. Solutein and Soluteout therefore represent
substrate-like and product-like states in reactions catalyzed
by passive transporters (e.g., Solutein # Soluteout) and
active transporters (e.g., Solutein þ ATP # Soluteout þ
ADP þ Pi; or, Solutein þ Gradient-State1 # Soluteout þ
Gradient-State2). The now classical work by American
biochemist Ronald Kaback demonstrated how lactose
permease couples lactose transport to a transmembrane
proton gradient.
Another example of non-covalent catalysis is the Naþglucose symport system, which mechanochemically links
the energy stored in a transmembrane sodium gradient to
drive glucose uptake. This transporter operates by the same
random substrate addition mechanism as that observed with
enzymes like hexokinase and creative kinase.
Na+
G
+
PE▪X+
Na+
Glc
Glc
Catalyzed
Tout Na+
PE+ AD+T
Complex2
Complex1
PE+AT+ D
Tout
Tin Glc
Tout Glc
Na+
Tin Glc
Tout Glc
Tin
Na+
Tin Na+
Reaction Progress
FIGURE 1.5 Profilin catalysis of exchange of solution-phase ATP
with actin-bound ADP to form solution-phase ADP with actin-bound
ATP. Symbols used are: A, Actin, AD, Actin$ADP ¼ Substrate; AT,
Actin-ATP ¼ Product, PE, Profilin acting as an Enzyme; Complex1 ¼
ProfilinE$Actin$ADP ¼ Enzyme$Substrate Complex; Complex2 ¼
ProfilinE$Actin$ATP ¼ Enzyme$Product Complex. Note: Profilin
catalyzes physical exchange of the entire nucleotide molecule, and not
phosphoryl transfer.
Glc
Na+
Glc
Na+
Scheme 1.7
In Scheme 1.7, the isomerization of the central pathway
represents the conversion of the transporting enzyme from
its outside conformation Tout to its inside conformation Tin.
Only when the sodium ion and glucose sites are occupied
Chapter j 1 An Introduction to Enzyme Science
25
does the symporter operate. Note again that no covalent
bond-making/-breaking steps are involved. Binding of
sodium ion actually increases the affinity of enzyme for
glucose to such an extent that greatly favors the upper path
(Crane and Dorando, 1980).
Foldases are mechanoenzymes that catalyze ratelimiting steps along the folding pathway of a protein,
including the cis-trans isomerization of peptidyl-prolyl
bonds as well as the formation/isomerization of disulfide
bonds. Molecular chaperones (sometimes regarded to be
a specialized class of foldases) are highly conserved
conformation-isomerizing enzymes found in all living
systems. They facilitate folding by interacting with misfolded polypeptide chains, but they do NOT become part of
the final structure or alter the equilibrium poise of the
Proteinunfolded # Proteinfolded equilibrium. Among the bestcharacterized molecular chaperones are GroEL-GroES and
DnaK-DnaJ-GrpE systems that are found in the cytoplasm
of Escherichia coli. Other molecular chaperones include
Clp ATPases, HtpG and IbpA-IbpB.
As will be discussed in Chapter 12, non-covalent
substrate-like and product-like states are of paramount
importance in the action of mechanochemical enzymes (or
simply mechanoenzymes). These highly specialized
enzymes use chemical bond energy to perform work (i.e.,
generate a force F over a distance Dx). Chemical-tomechanical energy transduction is accomplished by means
of an affinity-modulated binding interaction, generally
using the Gibbs free energy of ATP (or GTP) hydrolysis to
control the strength of their binding to their metabolic target
(e.g., other enzymes, proteins, transported substances,
cytoskeletal and membrane components, as well as nucleic
acids). Although each mechanoenzyme has its distinctive
mechanistic features, the general scheme can be depicted as
follows:
StateS + Enz·ATP
StateS·Enz·ATP + H20
+
StateS·Enz-ATP
Proposed New Class: Energases – catalyze the transduction of chemical-bond energy into noncovalent interactions
that generate force and do work.
While instituting a new class represents a challenging
task – one involving upwards of 1,000 enzymatic activities,
those resisting such change ignore the obvious: enzyme
names and classes should account for the entire chemical
reaction – and not just the covalent chemical bonds.
Finally, although many of the enzymes described here
are relatively feeble catalysts (e.g., profilin’s rate enhancement e is only ~140–150), especially compared to other
enhancement factors of 1015, the phenomenon of catalysis
has nothing to do with the magnitude of rate enhancements.
If the uncatalyzed reaction (or reference reaction) is already
fairly rapid, the catalytic rate enhancement need not be great
for the catalyzed rate to proceed on a physiologically
meaningful time-scale. Natural selection provides a rationale for the attainment and maintenance of evolutionary
advantages. Mutations making an enzyme more efficient
than necessary (i.e., ‘‘over-perfection’’) offer the cell no
durable advantage, and may even prove to be deleterious
(e.g., by allowing undesirable accumulation of pathway
intermediates). Simply put, an enzyme need only be as good
a catalyst as Nature demands in the context of the overall
biochemical process.
+
StateS*·Enz·ADP·Pi·H
+
StateS*·Enz·ADP·Pi·H
StateP·Enz·ADP + Pi + H
StateP·Enz·ADP + ATP
StateP + Enz·ATP + ADP
StateS + ATP + H20
components are formed, remodeled, and degraded enzymatically. Endocytosis and organelle traffic, cell crawling,
signal transduction, and mitosis/meiosis are processes that
are taking on the appearance of the pathways of intermediary
metabolism. Even long-term potentiation, a neuronal
process lying at the root of our memory and consciousness, is
now known to depend on actin polymerization motors to
maintain and/or remodel dendritic spines into synapses.
Because these energy-driven, affinity-modulated mechanoenzymes must be distinguished from energy-dissipating
hydrolases (e.g., ‘‘ATPases’’ and ‘‘GTPases’’), Purich
(2001) indicated the need for an additional enzyme class:
+
StateP + ADP + Pi + H
Scheme 1.8
where the braces are used to indicate complexes, and the
asterisk indicates a conformationally energized species.
Note also the various states where the mechanical work can
be accomplished. The field of cell biology can be largely
regarded as structural metabolism, where the supramolecular
1.5 EXPLAINING THE EFFICIENCY
OF ENZYME CATALYSIS
Biochemists and chemists alike have struggled to explain
why enzyme catalysis is so extraordinarily fast. As stated by
Warshel et al. (2006), ‘‘the real puzzle is why the enzyme
reaction with the specific chemical groups (e.g., acids and
bases) is so much faster than the reaction with the same
groups in solution.’’
The efficiency of biological catalysis is in fact so great
that the best way to assess the efficiency is to compare the
free energies of activation DGact, which are by definition
proportional to ln(k). When comparing catalyzed and
uncatalyzed processes, it is also essential to compare the
activation energies for and enzyme-catalyzed reaction and
Enzyme Kinetics
26
50
ΔG++cat
ΔG++w,w
G (kcal/mol)
40
ΔG++p,w
30
20
10
0
1
2
3 4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
Reaction System
FIGURE 1.6 Activation free energies for representative enzymatic
reactions (DGcat), reference reactions operating by the same mechanism (DGp,w), as well as the actual mechanism in water (DGw,w). The
reactions are those catalyzed by: 1, ketosteroid isomerase; 2, aldose reductase; 3, carbonic anhydrase; 4, chorismate mutase; 5, trypsin; 6, haloalkane
delahogenase; 7, alkaline phosphatase; 8, Ras GTPase complexed to its
activating protein GAP; 9, triose phosphate isomerase; 10, acetylcholine
esterase; 11, lysozyme; 12, RNase (mono-ionic intermediate); 13, RNase
(di-ionic intermediate); 14, ATPase; 15, bacteriophage T7 DNA polymerase; 16, orotidine 59-monophosphate decarboxylase; 17, exonuclease
activity of DNA polymerase I (Klenow fragment); and 18, staphylococcal
nuclease. Figure and legend reproduced from Warshel et al. (2006) with
permission of the authors and publisher.
the corresponding reaction (i.e., the reference reaction) that
operate by the very same mechanism, rather than just the
same chemical reaction as it occurs in water. Figure 1.6
presents such a graph from Warshel et al. (2006) comparing
eighteen different reactions in terms of DGcat, the activation
energy for the enzymatic reactions, DGp,w, the activation
energy for the corresponding reference reactions operating
by the very same mechanism in water, as well as DGw,w, the
activation energy for the actual mechanism in water.
Among the explanations for such phenomenal efficiency
are: the use of binding energy to stabilize reaction transition
states, the catalysis-promoting role of electrostatics in
stabilizing transition states, the role of reactant approximation and orientation (including orbital steering) in
guiding substrate interactions with catalytic groups, the role
of low-barrier hydrogen bonds in stabilizing reaction transition states, the coordination of acidic and basic groups in
‘‘pushing’’ or ‘‘pulling’’ protons to and from reactants, the
role of the enzyme in destabilizing substrate ground states,
the formation of covalent intermediates in preserving group
transfer potential, the roles of metal ions as templates,
Lewis acids, and special redox states, as well as the catalytic
role of enzyme conformational dynamics, including
inherent force-sensing, force-managing and force-generating mechanisms.
While enzymes simply must decrease the activation
energy for the reactions they catalyze, determining exactly
how this is accomplished has stubbornly resisted quantitative explanations. Part of the answer is that enzymes are
most often catalytically processive, meaning that, beyond
some point in their respective catalytic cycles, they remain
tenaciously associated with their reaction intermediates
until catalysis is accomplished. Their active sites are also
highly flexible, facilely adapting to meet the needs for rapid
acid-base and/or electron transfer reactions. When
combined with their substrate, these active sites serve as
ideal ‘‘solvents’’ – at times aqueous protic solvents, and at
other times nonaqueous protic solvents, while always
stabilizing the succession of enzyme-bound intermediates
comprising a catalytic cycle.
As we shall see throughout this book, virtually all classes
of organic reactions observed in the chemical laboratory
have one or more enzyme counterparts. Much as the most
successful chemists, who exploit the laboratory to improve
the rates and yields of these reactions, enzymes have
exploited evolution to become highly effective catalysts. At
this point, they have developed highly effective mechanisms
that doubtlessly take fullest advantage of catalytic strategies
as described in Sections 1.5.1 through 1.5.11, but optimized
from start to finish for extreme efficiency. The late BritishAmerican chemist Jeremy Knowles adopted the title
‘‘Enzyme Catalysis: Not Different, Just Better’’ for his
cogent discussions of catalytic rate enhancement (Knowles,
1991). His view was that enzymes operate by highly perfected catalytic mechanisms that, with the exception of their
speed and specificity, resemble those explored for decades
by physical organic chemists. That said, the development of
a precise quantitative model for enzymatic rate enhancement, even for a single enzyme, remains an elusive goal.
While various explanations discussed below are based on
principles from physical organic chemistry, nearly all focus
on the stability of enzyme transition states and/or the
dynamic flexibility of enzymes.
1.5.1 Stabilization of Reaction Transition
States
Without specifying how, J. B. S. Haldane (1930) offered
the idea that enzymes lower the activation energy for
catalysis. Linus Pauling (1946; 1947) took the matter
further by attributing enhanced catalysis to an enzyme’s
ability to interact with and stabilize the reaction transition
state (a proposal now known as Transition-State
Stabilization).
+
EX+
+
Stabilize EX+
+
EX+
E+S
E+S
ES
ES
The idea was that each enzyme becomes structurally
complementary to the transition state, such that the
Chapter j 1 An Introduction to Enzyme Science
27
geometry, polarity, and electrostatic charge of the enzyme
and the transition-state configuration of the substrate are
mutually stabilizing. Pauling (1947) wrote:
From the standpoint of molecular structure and the
quantum mechanical theory of chemical reaction, the
only reasonable picture of catalytic activity of enzymes
is that which involves an active form of the surface of
the enzyme which is precisely complementary in structure
not to the substrate molecule itself, but rather to the
substrate molecule in a strained configuration corresponding to the ‘activated complex’ for the reaction catalyzed by
the enzyme: the substrate molecule is bound to enzyme,
and caused by forces of attraction to assume the strained
state which favors the chemical reaction – that is, the activation energy of the reaction is decreased by the enzyme to
an extent as to cause the reaction to proceed at an appreciably greater rate than it would in the absence of the
enzyme.
The key point is that the enzyme need not initially be
complementary to the transition state configuration. An
active site that accommodates a reaction transition state
would also tend to stabilize those forms of the substrate that
most closely resemble the transition state both geometrically and/or electronically. Transition-state stabilization
thus makes it easier for the substrate to reach and surmount
the transition-state, and the net effect should be greatly
enhanced chemical reactivity.
Note that little advantage would be gained if an enzyme
were to stabilize both the E$S and E$Xz equally, because the
activation energy would remain the same as that occurring
in the absence of the enzyme.
+
+
EX+
EX+
Stabilize
ES Only
E+S
E+S
+
E+S
ES
+
EX+
EX+
Stabilize
+
Stabilize+
EX+Only
ES & EX+
E+S
ES
ES
ES
As we shall see in Section 8.6, these ideas are also
consistent with the action of both naturally occurring and
synthetic enzyme inhibitors that are structurally analogous
to the reactant’s transition-state. By mimicking the transition state, these analogues can bind to an enzyme with
extraordinary affinity, simply because the enzyme need not
divert a great deal of its binding energy to rearrange the
analogue into a configuration resembling the reactant in its
transition state (Schramm, 2003; Wolfenden, 1969).
1.5.2 Electrostatic Stabilization
of Transition States
As the name implies, electrostatic catalysis is the consequence of the strong local Coulombic interactions that
stabilize ionic and polarized transitions states. The presence
of such charged groups actually makes the active site’s local
environment significantly more polar than water (Warshel
and Florián, 1998), allowing ionic transition states to be
stabilized by nearby fixed dipoles. The nucleophilic and
electrophilic properties of functional groups on the catalyst
and reactant are also increased by dehydration of the catalytic center. In addition, electrostatic attraction and charge
neutralization tend to release water from enzyme active
sites, thereby exerting a powerful activating effect on
nucleophilic reactions.
Note that stabilization of the very same transition state in
bulk water would require a substantial thermodynamic
penalty, referred to as a reorganization energy, for water
molecules to be arranged in a manner that stabilizes ionic
transition states. In enzymes, the ionic groups are preorganized by protein folding, such that the resulting facilitated catalysis is attended by a very small reorganization
energy. Folding of the enzyme creates a constellation of
positively and negatively charged functional groups that are
appropriately positioned for optimal catalysis. This concept
may be extended to include catalysis-promoting changes in
electrostatic interactions as reactants proceed through the
reaction cycle, including effects of conformational changes
and hence enzyme dynamics on electrostatic interactions
and vice versa.
Coulombic interactions mainly occur among acid and
base groups in the enzyme (as well as ionizable groups
with its substrate). Metal ions also play important roles in
electrostatic catalysis. In some cases, other permanently
charged side-chain groups (e.g., the guanidinium of arginine and the quaternary ammonium group of 3-methylhistidine) may contribute to electrostatic stabilization of
transition states. Another advantage of electrostatic effects
is that they are ‘‘tunable,’’ meaning that the local environment can alter the pKa values of acidic and basic
groups. For example, when placed into a hydrophobic
environment, acids tend to exhibit higher pKa values (i.e.,
formation of the carboxylate anion –COO is disfavored),
whereas bases tend to have lower pKa values (i.e.,
formation of cationic –NHþ
3 groups is disfavored). When
in the vicinity of a residue of like charge, acids likewise
tend to exhibit higher pKa values, whereas bases tend to
have lower pKa values. When in the vicinity of a residue of
opposite charge, acids likewise tend to exhibit lower pKa
values, whereas bases tend to have higher pKa values.
Finally, a-helices also have associated dipole moments
that can also exert electrostatic effects on active-site
functional groups (Hol, 1985). Knowles (1991) discussed
how one particularly well-aimed helix in triose phosphate
isomerase is trained on His-95, lowering the pKa value of
the latter from an unperturbed value of 6 to below 4.5.
Charge neutralization can also exert a strong desolvating
effect within active sites. Prior to neutralization, each
active-site cation and anion binds several water molecules,
Enzyme Kinetics
28
such that [Cation$(H2O)k]xþ þ [Cation$(H2O)l]y ¼
[Salt$(H2O)(kþl)m] þ mH2O.
Warshel et al. (2006) presented persuasive arguments
that the catalytic power of enzymes may well be almost
entirely the consequence of electrostatic stabilization of
the transition state. Among the many examples, two
classical cases are the lysozyme reaction, for which the
oxacarbenium ion is stabilized by nearby carboxyl
groups, and the chymotrypsin reaction, in which the
tetrahedral oxy-anion intermediate is stabilized by the
intrinsically electrostatic phenomenon of hydrogen
bonding.
O
OH
O
O
O
O
O
HO
HO
OH
The lysozyme mechanism was first analyzed computationally by Warshel and Levitt (1976), who were among the
earliest proponents of computer-based molecular modeling
to assess the origin of enzyme rate enhancements. Ideally,
one desires a quantum mechanical (QM) model defining all
the atoms in the reactant and catalyst. An inherent limitation
on QM calculations, however, is that the required computational time rises very steeply with increasing numbers of
atoms and electrons in the molecule(s) of interest, making
studies of entire enzyme-reactant interactions totally
unworkable. To maneuver around this limitation, Warshel
and Levitt (1976) pioneered a combined QM and molecular
mechanical (MM) approach, restricting the quantum
chemical description to the reaction center, while relying on
a computationally efficient classical treatment for the
remainder of the molecule. Based on their QM/MM results
with lysozyme, Warshel and Levitt (1976) suggested that
the positive charge developing on the C-1 carbon of the
glucopyranosyl residue would be stabilized by the adjacent,
electron-rich ring oxygen and the charge-neutralizing
effects of nearby glutamate and aspartate. Importantly, Sun,
Liao and Remington (1989) used classical electrodynamics
to find that C–O bond breakage and the consequent charge
separation is promoted by a large electrostatic field across
lysozyme’s active-site cleft, created in part by a very
asymmetric distribution of charged residues on the enzyme
surface. That other lysozymes of unrelated primary
sequence have similarly distributed charged residues and
electric fields suggests the generality of electrostatic stabilization (~9 kcal/mol) as the basis for catalytic rate
enhancement in lysozyme.
Because the hydrogen bond is primarily an acid-base
neutralization, electrostatic catalysis also explains the role
of hydrogen bonding in fostering catalysis. Two hydrogen
bonds stabilizing the oxyanionic tetrahedral intermediate in
chymotrypsin catalysis can contribute 7–8 kcal/mol of
transition-state stabilization, resulting in considerable
catalytic rate enhancement.
1.5.3. Intrinsic Binding Energy
Binding energy effects arise from the sum total of favorable
non-covalent interactions between an enzyme and its
substrate(s), including a substantial contribution from van
der Waals interactions associated with structural complementarity of the enzyme and its substrate as well as desolvation. The favorable enthalpy of substrate binding is
thought to overcome the unfavorable entropy associated
with bringing two (or more) molecules together. Once
formed, the E$S complex allows the catalysis to be effectively an intramolecular process.
Jencks (1975) suggested that enzymes gain great
advantage over ordinary catalysts by managing the energy
of binding interactions to orient substrates relative to each
other and with respect to catalytic groups within the active
site. Page and Jencks (1971) showed that the loss in
entropy in going from a bimolecular to a unimolecular
reaction (i.e., E þ S # E$S) results in the loss of translational, rotational and vibrational degrees of freedom, thus
accounting for around 108 of the rate enhancement
observed in enzyme-catalyzed reactions. Based on results
of their computer modeling of subtilisin interactions with
model substrates, Villá et al. (2000) reached a completely
different conclusion – namely that the contribution of DSz
to DGz is much smaller than previously thought. They
suggest that this is true because: (a) many of the motions
that are free in the reactant state of the reference reaction
are also free at the transition state; (b) the binding to the
enzyme does not completely freeze the motion of the
reacting fragments, so that DSz in the enzyme is not zero;
and (c) the binding entropy is not necessarily equal to
DSzwater.
1.5.4 Reacting Group Approximation,
Orientation and Orbital Steering
Substrate binding to the active site promotes catalysis:
(a) by converting multi-substrate reactions from bimolecular rate processes to what essentially becomes a unimolecular rate process; (b) by increasing the effective local
concentration of reactants with respect to each other; and (c)
by arranging and orienting reactant functional groups with
respect to each other. In most instances, intramolecular
reactions occur at much faster rates than corresponding
bimolecular reactions, and both proximity and orientation
can increase the effective local concentrations of reactants.
As discussed in Chapter 3, this behavior is related in part to
the brief lifetime of collision and encounter complexes,
Chapter j 1 An Introduction to Enzyme Science
leaving fleetingly short times for productive chemical
reactions to occur. Proper placement and orientation of
reactive groups is also recognized to play a major role in
catalytic rate enhancement through stereo-electronic assistance, wherein reactants are arranged for maximal reactivity. Of course, orientation comes at a price (i.e., often
manifested by a decrease in entropy) which must be offset
by some other favorable catalyst–substrate binding interactions in regions that are immediately adjacent to the bonds
that are broken and/or made during catalysis.
Inspired by the then obvious success of the conservationof-orbital-symmetry rules (Woodward and Hoffmann,
1970) in explaining reactivity, Storm and Koshland (1970)
proposed and Dafforn and Koshland (1971; 1973) suggested
that enzymes may promote catalysis by precisely aligning
(steering) the molecular orbitals of their substrates. In the
earliest versions of Orbital Steering, enzyme-enforced
constraints on molecular orbital alignment were viewed as
so restrictive as to be physically unrealistic, and the
proposal was roundly dismissed on the basis of the anticipated high thermodynamic penalty for extremely precise
orbital alignment and the weak dependence of force
constants on slight changes in bond angle (Bruice, 1972;
Jencks and Page, 1974).
Noting that the contribution of orbital steering to
catalytic rate enhancement cannot be quantified in the
absence of an accurate means for correlating structural
interactions and catalytic enhancement, Scott (2001)
argued that orbital steering may explain aspects of RNA
catalysis. For ribozymes, orbital steering appears to be
fortuitously uncoupled from conformational, distance and
orientation effects. During hammerhead ribozyme catalysis, two conformational changes appear to align the
orbitals of reacting atoms, and Scott (2001) suggested that
each of these two conformational changes is likely to
provide rate enhancement 3 of ~1,000. With an overall
rate enhancement of 106 that is solely attributable to
orbital steering, Scott (2001) suggested that orbital steering is a significant factor in the catalysis of ribozymes and
protein enzymes.
For additional comments on orbital steering and its
implications, the interested readers should consult valuable
reviews by Hackney (1990) and Mesecar, Stoddard and
Koshland (1997).
1.5.5 Reactant State Destabilization
In this case, the enzyme strains or distorts the substrate
while still in its ground-state, making the reactant(s) less
stable and thereby lowering the energy difference (indicated
by red arrows) between strained reactant(s) and transition
state. (Note: The terms ground-state destabilization (GSD)
and reactant-state destabilization (RSD) are interchangeable.) Some form of reactant distortion, bond strain, bond
29
polarization, E$S conformational change, and/or electrostatic effect would presumably be required.
EX
+
Stabilize EX+
+
EX+Only
Form Unstable
ES Complex ES
E+S
ES
E+S
Perhaps the best-known version of ground-state destabilization is the Circe Effect. Jencks (1969) suggested that
highly favorable substrate binding interactions in a substrate’s
nonreactive region may facilitate catalysis by forcing the
substrate’s reaction center into a destabilizing environment.
The Circe Effect is viewed as using substrate binding energy
to help reacting groups to approach the transition state. The
name of this effect derives from the mythic Greek enchantress
Circe whose sweet songs beguiled passersby to her island,
where they were then transformed through the action of her
various spells and potions.
Bruice (2002) suggested that an enzyme positions its
substrate(s) in a conformation, such that thermal fluctuations allow that conformation to easily surmount the barrier
to reaction. The basic idea is that for covalent bond
formation, reacting atoms of substrate and enzyme must
first come together within a suitable reaction distance (say
3–4 Å) and approach angle (say 5–10 ), such that suitably
rearranged and highly reactive ground-states, termed NearAttack-Conformers (NACs), would thereby accelerate
catalysis. In this explanation, the enzyme might bind
strongly to a transition-state structure, but this binding
energy is not thought to be released specifically to speed
the reaction (Luo and Bruice, 2004; Torres, Schiott and
Bruice, 1999). Except for the speculative role of anticorrelated motions of proximal residues in destabilizing the
substrate (Luo and Bruice, 2004), the notion of NACs
merely restates the obvious, in that reactant-state destabilization is merely an alternative description of transitionstate stabilization. Warshel et al. (2006) noted that, if both
the reactant state (RS) and transition state (TS) for an
enzyme-catalyzed reaction were to have similar charge
distributions, the same preorganization effects are apt to
stabilize the RS and TS, leading to an apparent NAC effect
by making the RS structure closer to that of the TS. They
thus argue that the so-called NAC effect is an expected
result of the TS stabilization rather than the underlying
cause of catalysis.
In a sense, if Near-Attack-Conformers are viewed as
enzyme-stabilized ‘‘pre-transition-state’’ structures facing
only a modest barrier to reaction, they might just as well be
thought of as part of an ensemble of enzyme-stabilized
transition states. Indeed, the smaller the barrier to reaction,
the more like a stabilized transition state would be an NAC.
Enzyme Kinetics
30
1.5.6 Acid/Base Catalysis
As discussed in Chapter 2, formation of formal cationic or
anionic species, each respectively possessing fully developed electronic charges on the electron deficient or electronrich atom, is a highly improbable event that necessarily
results in a high DEact for reaction. Acids and bases often
improve reactivity, and transfer of a proton (to the reactant by
an active-site acid and from the reactant by an active-site
base) has the effect of lowering the energy of the transition
state, thereby reducing the activation energy DEact. An even
greater enhancement is attained by the coordinated action of
an acid and base, as in the case of an active-site base attacking
a carbonyl, with attendant protonation by an active-site acid.
A
H
O
R
B:
S
R
Scheme 1.9
The virtually simultaneous action of nucleophilic attack
and protonation (Scheme 1.9) requires additional structural
organization within the active site, requiring appropriate
orientation of enzyme functional groups. If the energy
penalty for pre-organization is ‘‘paid’’ upon folding of the
nascent protein, much as suggested for electrostatic catalysis, then acid/base catalysis requires no additional energy
penalty for functional group orientation.
Numerous observations confirm that complete proton
transfer need not occur. In fact, significant advantages
accrue when a Brønsted acid partially donates a proton to
(or when a Brønsted base partially abstracts a proton from)
a reaction center (see Section 7.3.9: Brønsted Theory
Explains Important Aspects of Acid/Base Catalysis).
Finally, because the equilibrium between the active-site
base and its conjugate acid (or the active-site acid and its
conjugate base) is coupled to the catalytic cycle, enzyme
activity frequently displays a pH-dependence. Although
steady-state kinetics is effective in discerning the pH
dependencies for Km, Vm, or Vm/Km, the pH-dependence of
elementary reactions rates is far more revealing. This is true
because steady-state parameters like Km, Vm, or Vm/Km are
complex collections of elementary reaction rate constants,
whereas fast kinetic studies directly establish the pH
dependence of individual elementary rate constants.
intermediates confirms that significant advantages must be
gained from their formation. Enzymes organize covalent
intermediate formation and turnover into discrete stages:
first, there is a nucleophilic stage, in which a catalytic
functional group attacks the substrate to form a covalent
bond; second, electrons are withdrawn by the now electrophilic catalyst; and third, rupture of the covalent bond
permits further reaction and regenerates the enzyme-based
nucleophile. The latter is typically the functional group of
a lysine, histidine, cysteine, aspartate, glutamate, and serine
residue within an enzyme’s active site. Many coenzymes
(e.g., pyridoxal 5-phosphate, biotin, lipoamide, thiamin
diphosphate, tetrahydrobiopterin, and even NADþ and FAD)
also play essential roles in forming covalent intermediates.
As discussed by Jencks (1963; 1969), formation of a covalent
intermediate per se is insufficient for highly effective covalent
catalysis: beyond reacting rapidly, the active-site nucleophile
must yield a product that is itself highly reactive. He also
asserted that the chief advantage of enzymatic covalent catalysis
is that reaction mechanism can be organized in a manner that
manages entropy changes (Jencks, 1975) while maintaining the
group transfer potential of substrate-derived moieties (e.g.,
phosphoryl groups, amino groups, nucleotidyl groups, etc.).
That covalent catalysis requires a highly reactive activesite nucleophile is well illustrated by the following two
reactions. In the case of bacterial acetoacetate decarboxylase
(Reaction: Acetoacetate # Acetone þ CO2), the group
headed by Frank Westheimer at Harvard University
demonstrated that the enzyme exploits its surprisingly acidic
e-amino group (pKa z 6.5), which is displaced by some four
pH units from that of a typical lysine side-chain amino group.
O
H3C
C
O
H2
C
H3C
C
O
Covalent catalysis refers to any catalytic rate enhancement
gained from transient formation of covalent reaction intermediates. That thousands of enzymes form covalent
H2
C
O
C
O
NH
H 2N
Enz
Enz
C O2
H 3C
H2
C
C
H
O
H3C
C
O
NH
C
C H2
N
Enz
H
Enz
Enz
H3C
C
NH
C H3
NH2
OH
H3C
C
C H3
O
Enz
1.5.7 Covalent Catalysis
C
Scheme 1.10
Shown in Scheme 1.10 is the likely mechanism showing
how the formation of imine and eneamine intermediates
Chapter j 1 An Introduction to Enzyme Science
31
organizes stepwise decarboxylation (see Section 7.3.4 for
a detailed discussion of this enzyme mechanism). Likewise,
the research group headed by Daniel Santi at UC San
Francisco demonstrated that thymidylate synthase (Reaction: dUMP þ Methylenetetrahydrofolate (CH2–H4Folate)
# dTMP þ Dihydrofolate (H2Folate)) exploits covalent
catalysis to activate dUMP for subsequent substitution
(Carreras and Santi, 1995). After forming a reversible
ternary complex with its substrates, this synthase directs the
nucleophilic attack of its active-site thiol on C-6 of dUMP,
converting C-5 into a nucleophilic enol(ate) intermediate.
Subsequent covalent bond formation ensues between that
site and the one-carbon unit (at C-11) of CH2–H4Folate,
itself having been activated by formation of an N-5 iminium
ion. Proton abstraction from the second key intermediate
and b-elimination of H4Folate yields the exocyclic
methylene intermediate. Hydride transfer from noncovalently bound H4Folate to the exocyclic methylene
intermediate is followed by b-elimination of the enzyme,
producing dTMP and H2Folate as well as regenerating the
original active enzyme.
An added benefit of covalent catalysis is that reactive
intermediates can be shuttled from one active site to another
in multi-enzyme complexes. One example is transcarboxylase (Reaction: Methylmalonyl-CoA þ Pyruvate #
Propionyl-CoA þ Oxaloacetate), which catalyzes a multisite Ping Pong mechanism (Northrop, 1969).
S
H
N
O2C N
ENZYME
NH
O
O
The transferred carboxyl group (shown in red) is carried
from one active site (First Half-Reaction: Enz þ Methylmalonyl-CoA # Propionyl-CoA þ Enz–CO2) to a second
active site (Second Half-Reaction: Enz–CO2 þ Pyruvate
# Oxaloacetate þ Enz) by means of a long arm consisting
of a biotin cofactor (blue) covalently tethered to an
3-amino group of transcarboxylase lysine residue (black).
Another example is pyruvate dehydrogenase (PDH),
a multi-enzyme system that uses five cofactors: NADþ,
coenzyme A, thiamin diphosphate (TDP), lipoamide, and
FAD. PDH catalyzes the overall reaction of pyruvate with
NADþ and coenzyme A to produce acetyl-CoA, NADH,
and CO2. PDH first catalyzes the TDP-dependent reaction
of pyruvate with lipoamide to form S-acetyl-dihydrolipoamide and CO2. Dihydrolipoamide S-acetyltransferase next catalyzes the reaction of S-acetyldihydrolipoamide with coenzyme A to produce dihydrolipoamide and acetyl-CoA. Then the FAD-dependent
dihydrolipoamide dehydrogenase uses its active site to
catalyze the reaction of dihydrolipoamide with NADþ to
produce lipoamide and NADH. Without the intervening
synthesis of covalent intermediates, pyruvate dehydrogenase catalysis would presumably require additional steps as
well as the release of reactive intermediates.
Finally, in some enzyme-catalyzed reactions, formation of covalent intermediates also affords the opportunity to control overall reaction stereochemistry. Two SN2
reactions are needed to form and transfer a reactive
covalent intermediate, a scheme that results in overall
retention of configuration. With direct in-line transfer,
however, only one SN2 reaction is needed, resulting in
overall inversion. Interestingly, for SN1 mechanisms, the
stereochemical outcome depends on how the carbenium
ion intermediate is intercepted. Because subsequent
enzyme-catalyzed reactions within a metabolic pathway
are often stereospecific, the stereochemical course of
a preceding enzyme must be maintained, and a mechanism requiring covalent catalysis successfully fulfills this
requirement.
1.5.8 Transition-State Stabilization
by Low-Barrier Hydrogen Bonds
A special type of transition-state stabilization, first suggested by Schowen (1988) and promoted by Cleland and
Kreevoy (1994), concerns the possibility that the protected
interior of certain active sites may favor formation of strong
hydrogen bonds, known as low-barrier hydrogen bonds
(LBHBs). Unlike most other hydrogen bonds, which have
2.9–3.3 Å distance between electronegative atoms, the
bond-length of low-barrier H-bonds is less than 2.5 Å.
Neutron diffraction experiments on crystalline compounds
containing LBHBs indicate that the shared proton is
diffusely distributed around the bond’s midpoint, a finding
suggesting that LBHBs exhibit covalent nature (see also
Section 2.2.3).
Cleland, Frey and Gerlt (1998) suggested that lowbarrier hydrogen bonds may contribute upwards of five
orders of magnitude in rate acceleration in any enzymatic
reaction involving proton transfer from a general acid or to
a base. Their argument goes as follows: LBHBs form when
the atoms sharing the proton have identical pKa values; so
any equalization of their pKa values should enhance H-bond
overlap, thus stabilizing the transition state and promoting
catalysis. Cleland and Kreevoy (1994) suggested that
LBHBs may provide up to 10–20 kcal/mol of transitionstate stabilization; however, model studies on LBHBs put
the value at 4–5 kcal/mol in dimethyl sulfoxide and 3–6
kcal/mol in tetrahydrofuran (Shan, Loh and Herschlag,
1996). Usher et al. (1994) estimated the value to be nearer to
2 kcal/mol, a value that comports with mutagenesis data
(Fersht, 1987). Shurki et al. (2002) also questioned whether
LBHBs can account for catalytic rate enhancements
observed with protein enzymes. Paradoxically, Warshel
et al. (2006) argue that, when consistently defined,
Enzyme Kinetics
32
low-barrier hydrogen bonds are more apt to exert an anticatalytic effect.
1.5.9 Catalytic Facilitation by Metal Ions
Although enzyme- and substrate-bound metal ions exert
powerful electrostatic stabilization of transition states,
metal ions are known to facilitate catalysis in many other
ways. By taking advantage of the well-defined geometric
arrangement of their inner-coordination spheres, transition
metal ions often serve as templates that hold and orient
reactive molecules during one or more phases of the catalytic reaction cycle. Metal ions are also highly versatile
Lewis acids (i.e., electron-pair acceptors) that can alter the
reactivity of acidic and basic functional groups. Metal ions
alter the pKas of bound substrates as well as bound water,
thereby improving their tendency to react. Metal ions also
transiently switch oxidation states during catalysis, and in
some cases, they achieve unusually reactive higher oxidation states. Ca2þ and Mg2þ also bind to ATP4 to form
CaATP2 and MgATP2, thereby activating the latter
toward nucleophilic agents (see Section 2.5: Metal Ions in
Enzyme Active Sites).
1.5.10 Promotion of Catalysis via Enzyme
Conformational Flexibility
In seeking to summarize the mounting evidence for a role of
protein dynamics in enzyme catalysis, Hammes (2002)
offered the following comments:
When a substrate binds to an enzyme, it becomes an integral part of the macromolecule. The subsequent dynamics
of the macromolecular conformational changes are then
the catalytic process itself. This view of catalysis means
that the making and breaking of non-covalent bonds within
the structure are part of the catalytic process, and that these
events can occur close to and far from the active site. The
advantage of having hundreds of intramolecular interactions dynamically involved in catalysis is that the energetics of the reaction can be easily manipulated to
produce catalysis, and extremely fine-tuning is provided
by hundreds of intramolecular interactions. This mechanism could be viewed as a ‘gear shift’ mechanism: the
conformational transitions are analogous to shifting gears,
and the interactions between the enzyme and substrate
correspond to the gear coupling mechanism. Asking what
‘drives’ the reaction is not terribly meaningful, as the
essence of cooperative processes is that many events are
occurring essentially simultaneously.
Hammes’ cogent comments are tantamount to saying
that an enzyme creates a dynamic catalytic environment,
one that promotes the trajectory of substrate to product by
way of one or more reaction intermediates. Much as wellrehearsed actors cue each other, a succession of structural
cues, each created as the enzyme and reactant proceed
step-by-step through the catalytic cycle. As indicated in
Fig. 1.4, the same catalytic histidine residue acts as a: (a)
general base by accepting the proton from the catalytic
serine, thereby activating the latter’s nucleophilicity; (b)
general acid by donating a proton to the nitrogen on the
leaving group; and (c) general base that deprotonates and
activates the ‘‘hydrolytic’’ water.
In the context of Hammes’ comments, it seems clear that
conformational changes in chymotrypsin trigger changes in
catalytic group reactivity and vice versa. Radisky et al.
(2006), for example, found that atomic-resolution structures
of acyl-trypsin and enzyme-bound tetrahedral intermediate
analogue, along with earlier structures for the Michaelis
complex, provide evidence of subtle active-site adjustments
favoring the forward progress of the acylation reaction. It
should be emphasized that the energetics at each reaction
stage can be finely tuned to facilitate catalysis without
violating the constraint that an enzyme cannot alter the
overall reaction’s equilibrium poise.
Mutual cuing between catalyst and reactant also fits with
action–reaction principles of classical mechanics. Therefore, although E$Xz is almost universally employed to
represent an enzyme-bound transition state, (E$X)z is
perhaps a more appropriate indicator that both the catalyst
and substrate are mutually altered as they proceed through
each catalytic cycle. This effect represents an example of
thermodynamic reciprocity (i.e., a catalyst cannot affect the
reactant without the reactant affecting the catalyst). Use of
E$Xz leaves a mistaken impression that only the substrate
reaches the activated complex or transition-state configuration. If this were the case, the intimacy of motions within
an enzyme-substrate would be ignored. Writing the overall
transition state as (E$X)z implies that enzyme and substrate
jointly achieve transition-state intermediacy, a process
requiring simultaneous motions in reactant and enzyme.
Another way of explaining the comparatively large size
of enzymes is that catalysis is a complex, multi-step process
requiring an active-site environment that optimally stabilizes multiple transition states, each associated with its own
step. Conformational flexibility is apt to be a hallmark of
effective multistage reaction catalysis and even suggests
why protein enzymes are apt to be more highly perfected
than nucleic acid enzymes. Benkovic, Hammes and
Hammes-Schiffer (2008) suggested that:
Enzyme mechanisms should be viewed as catalytic
networks with multiple conformations that occur serially
and in parallel in the mechanism. These coupled ensembles
of conformations require a multi-dimensional standard
free-energy surface that is very rugged, containing multiple
minima and transition states.
These features are shown in Fig. 1.7. As considered in
Section 12.3, this concept was anticipated in the derivation of
a single-molecule Michaelis-Menten equation by Kou et al.
(2005) who present a virtually identical view of an enzyme
Chapter j 1 An Introduction to Enzyme Science
33
1.5.11 Promotion of Catalysis via ForceSensing and Force-Gated Mechanisms
rm
ati
on
s
G°
oor
din
mb
le
nC
ate
E+P
se
ctio
En
Rea
Co
nfo
E+S
FIGURE 1.7 Schematic representation of the standard free-energy
landscape for a catalytic network of an enzyme reaction. The catalytic
process is viewed as proceeding through a network consisting of a multitude of conformations and numerous catalytic reaction cycles, each written
horizontally as a reaction path (e.g., E1 # A1 # B1 # # P1 # E1;
E2 # A2 # B2 # # P2 # E2; and E3 # A3 # B3 # # P3 #
E3, etc.), where each species is connected vertically to its corresponding
conformer (e.g., E1 # E2 # E3 # # En–1 # En). The result is
a network similar to that shown in Scheme 12.5 describing the results of
single-molecular enzyme kinetic data. For simplicity, only one substrate
S and one product P are shown. Note that enzyme conformational changes
actually occur along both axes: (a) those changes along the reaction coordinate axis correspond to the environmental reorganization facilitating chemical reaction; and (b) those changes occurring along the ‘‘ensemble
conformations’’-axis represent the ensembles of configurations existing at
all stages along the reaction coordinate. Therefore, a plane parallel to the
axis labeled ensemble conformations bisects this catalytic ‘‘mountain
range’’ along the red mountaintop, with reactants E þ S are on one side
of the plane and the products E þ P on the other. This free energy landscape
thus illustrates the multiple populations of conformations, intermediates,
and transition states. Strong coupling can occur between the reaction coordinates and the conformation ensembles (i.e., the reaction paths can slide
along and between both coordinates). For real enzymes, the number of
maxima and minima along the coordinates is expected to be greater than
shown. The dominant catalytic pathways will be altered by external conditions and protein mutations. Figure (originally created by S. J. Edwards)
and legend adapted from Benkovic, Hammes and Hammes Schiffer
(2008) with permission of the authors and the publisher.
operating by a catalytic network with multiple conformations
(see also Section 3.8: Transition State Theory).
Finally, although some nucleic acids serve as biological
catalysts, they are feeble in comparison to protein enzymes
(Purich, 2005). One may therefore speculate that the far
greater conformational flexibility of proteins and their
consequentially higher catalytic efficiency may have been
major driving forces in the early evolution away from
nucleic acid-based catalysts in favor of the far more versatile protein-based catalysts. Organisms managing to catalyze a reaction much faster than a rival should have enjoyed
a substantial advantage. Retention of some roles for catalytic RNA also suggests that rate enhancement may not be
as important for the catalysis of certain reactions.
As scientists, we should keep an open mind as to the
possibility that we have been trapped into thinking that
enzyme chemistry must operate by mechanisms that
resemble those for gas-phase and solution-phase organochemical reactions. The chemistry within enzyme active
sites may eventually prove to be fundamentally different.
All the enzymes that are known to catalyze some 10,000
to 20,000 different reactions in living organisms
constitute an infinitesimally small subset of 20500 possible
polypeptides of molecular masses of 50 kDa or less. What
may distinguish these 10,000 to 20,000 enzymes as the
rarest of the rare among those 20500 polypeptides is that
each has its respective reaction trajectory already programmed into its conformationally compliant structure. If
this unique catalytic choreography avoids the nonproductive
molecular configurations inevitably made in gas-phase and
solution-phase reactions, an enzyme may not require
a significant energy input to populate productive configurations. For example, the observation that heating gas-phase
and solution-phase reactants increases molecular agitation
and generally enhances reactivity is thought to be the
consequence of populating high-lying transition-state
configurations needed to convert a reactant into its product.
Even so, heating of reactants also produces countless
nonproductive configurations, thereby greatly limiting the
fraction of molecules that are appropriately oriented. Thus,
while we now think in terms of reaction coordinate
diagrams resembling those for solution-phase models,
enzymes may accelerate reactions in ways that are beyond
our reckoning, simply because we may be incorrectly
perpetuating the notion that enzyme mechanisms are ‘‘not
different, just better.’’ In carrying out covalent bond transformations, most enzymes may be acting in a manner that is
functionally complementary to the action of mechanoenzymes, meaning that they are programmed to make
directed motions via precise noncovalent bond rearrangements of the active site, and perhaps even the entire protein,
so as to convert the covalent bonds of the substrate into the
covalent bonds of the product. Simply put, although
enzymes undergo the same types of reactions and also
likewise form many of the same types of intermediates as
those observed in cognate gas-phase and solution-phase
organochemical, enzymes may not be confined by the rules
of physical organic chemistry, at least those rules gleaned
from studies of corresponding gas-phase and solution-phase
reactions.
There is reasonably general recognition that no single
property of an enzyme likely to underlie the origin of enzyme
rate enhancements and that each enzyme may exploit
more than one in the course of its catalysis. What becomes
evident is that many of the above ideas converge, if one
conceives of all enzymes – not just mechanoenzymes – as
34
force-generating and force-sensing molecular machines that
are exquisitely well designed to: (a) recognize and bind
specific substrates; (b) avoid unduly tight binding interactions with the substrate as well as catalytic cycle intermediates and reaction products; (c) bring about
conformational changes that continually re-position activesite groups as the catalytic cycle proceeds; and (d) promote
enzyme evolution and perfection by adjusting the energetic
landscape to create alternative mechanochemical pathways
for catalysis. In this way, some steps within a catalytic
reaction cycle may be viewed as force-gated conformational
changes that constantly readjust catalytic determinants
within the active-site to optimize the local push-pull force
balance between the enzyme and various forms of bound
reactant. All reaction coordinate diagrams plot DG (or
change in potential energy DU) on the ordinate versus
‘‘Reaction Progress’’ (often indicated as some inter-atomic
distance, say d, representing a bond making or bondbreaking event) on the abscissa. The slope DG/Dd (or DU/
Dd) corresponds to a pushing or pulling force F that is
mutually experienced by enzyme and its bound reactant(s) as
they jointly approach and surmount the transition state.
Such ideas also fit with the universal occurrence of
domains and motifs that are connected by hinges and joints,
where forces can be localized and/or managed (Williams,
1993). In this respect, an enzyme’s mechanochemical
properties appear to be a natural complement to its bondbreaking/making properties, as illustrated by the capacity of
ATP synthase to use ATP hydrolysis to drive the conformational changes that energize transmembrane proton
gradients or that use the latter to drive ATP synthesis
(Purich, 2001).
1.6 PROSPECTS FOR ENZYME SCIENCE
Predicting the likely direction that a scientific field will
take is an inherently hazardous enterprise, mainly because
field-changing intellectual realization occurs in bursts and
often exploits completely unanticipated opportunities. A
seemingly insignificant advance in one scientific discipline
may also trigger a breakthrough in another field. What is
self-evident is that enzymes have been bestowed with
a special status in the chemical sciences, and for nearly
two centuries, the chemical, biochemical, and physiologic
actions of enzymes have continually piqued the intellectual curiosity of highly creative individuals. By enriching
our understanding of enzymes and the physiologic
behavior, many enzymologists have even earned Nobel
Prizes (Table 1.4).
One may therefore assert that enzyme science will
surely enjoy a brilliant future, and it’s safe to assume that
this intellectually stimulating, and yet immensely practical, enterprise will doubtlessly prosper from the development of new kinetic approaches. The following
Enzyme Kinetics
sections describe areas where sustained inquiry is apt to
reap great rewards.
1.6.1 We Need Better Methods for Analyzing
Enzyme Dynamics to Understand the
Detailed Mutual Changes in Both Substrate
and Enzyme During Catalysis
As noted earlier in this chapter, the distinction between
chemical kinetics and chemical dynamics is that the former
focuses on the measurement of reactivity (i.e., reaction
rates) with an emphasis on bond-making/breaking mechanisms of chemical transformations, whereas the latter refers
to the atomic and molecular motions that influence reactivity and stability. While chemical intuition guides the
notion that internal enzyme flexibility is essential for
activity, the nature of catalytic motions is poorly understood. A longstanding question about biological catalysis
concerns the functional coupling of reactant motions to the
enzyme’s local conformational dynamics in various Enzyme$Substrate, Enzyme$Intermediate, and Enzyme$Product
complexes. The great speed of catalysis has been a major
obstacle for ‘‘on-the-fly’’ analysis of conformational
dynamics. Because the time-scale of each catalytic reaction
cycle sets the longest lifetime of any intermediate, an
enzymic reaction proceeding at a rate of 5,000 cycles/s has
a 0.2 millisec catalytic cycle-time, one that is too short for
most techniques that can detect individual residue sidechain motions.
There is in fact mounting evidence that protein dynamics
may play a central role in enzymatic catalysis, well beyond
the standard models of loop motions that help to hold
substrate(s) within a desolvated active site (Hentzler-Wildman and Kern, 2007). Directed motions of the enzyme per
se may be coupled to the catalytic mechanism, especially in
those cases where hydrogen tunneling seems to be operating
(Basran, Sutcliff and Scrutton, 1999). The basic idea is that
rate-promoting vibrations are intrinsic motions of the
protein catalyst that form a dynamic matrix surrounding
the substrate, and that these vibrational modes can alter the
geometry of the bonding barrier(s) to chemical reaction.
When viewed from this perspective, the defining nature of
a promoting vibration is to be found in the nature of the
coupling of that protein matrix motion to the reaction
coordinate (Caratzoulas, Mincer and Schwartz, 2002).
Antoniou et al. (2002) described how a catalysis-promoting
vibration within the enzyme may be coupled to a vibrational
mode of a reactant proceeding along the reaction coordinate. Their view is that evolution created a protein structure
that moves in such a way that lowers and narrows the barrier
to reaction. This lowering of the barrier is not merely
a statistical lowering of a potential of mean force through
the release of binding energy; rather, the enzyme is believed
to use highly directed energy in the form of a vibration
Chapter j 1 An Introduction to Enzyme Science
acting in a specific direction. It is believed that ratepromoting vibrations within protein catalysts have
150-cm1 frequencies, corresponding to vibrations on the
sub-picosecond time-scale. Because enzyme catalysis
occurs with frequencies of 104–107 s1, there is an unexplained disparity in vibrational and catalytic time-scales.
Many hundreds of thousands of these rate promoting
vibrations occur over the time needed for a single catalytic
round. Because vibrational energy obeys the Boltzmann
distribution (see Section 3.6: Thermal Energy: The Boltzmann Distribution Law), it’s possible that a rare (and hence
substantially more energetic) vibration may be needed to
trigger catalysis. Finally, Caserta and Cervigni (1974)
offered a more rudimentary suggestion that nonetheless
postulated electron induced, selective amplification of lowfrequency vibrational waves in the enzyme, such that these
vibrations are coupled to a susceptible region of the
substrate, with consequential lowering of the activation
energy.
Although hydrogen-deuterium and disulfide-trapping
techniques can clearly detect 15-Å protein motions on
the millisecond time-scale (Careaga and Falke, 1992;
Englander and Kallenbach, 1983; Falke and Koshland,
1987; Huyghues-Despointes et al., 2001), these methods
are uninformative about faster processes. For example, the
hydrogen deuterium exchange technique, which quantifies
the time-course for the release of protons bound up in
a-helix and b-sheet structures, is incapable of providing
such information on a sub-millisecond time-scale. Of
particular interest is whether picosecond and nanosecond
time-scale structural fluctuations are coupled to the
structural changes associated with the catalytic ratelimiting step, the latter typically occurring on the microsecond-to-millisecond time-scale (Daniel et al., 1999). An
important question is whether the fast motions need to be
anharmonic, such that picosecond-to-nanosecond motions
in the protein may be needed to permit slower microsecond millisecond dynamics across the highest-energy
reaction barrier.
The first parallel comparison of the activity and
dynamics of glutamate dehydrogenase (GDH), as probed
picosecond time-scale motions, showed no deviation from
Arrhenius behavior through the dynamical transition
(Daniel et al., 1998). The experiments were performed in
a 70% vol/vol methanol/water cryosolvent in which the
enzyme is active and stable. For the thermophilic microbial
GDH operating near 350 K, the turnover number of the
enzyme is ~1500 s1 at ~350 K, and in fully deuterated
cryosolvent at 220 K, the turnover number is ~0.01 s1.
Their results indicated that over the 190–220 K temperature
range, the enzyme’s rate-limiting step(s) is(are) unaffected
by picosecond protein motions. To extend the time-scale
problem, Daniel et al. (1999) used advanced neutron scattering spectrometers to compare the temperature dependence of GDH activity and dynamics. The IN6 spectrometer
35
probed motions on time-scales shorter than ~100 ps, and the
IN16 spectrometer extended the time-scale to ~5 ns. Their
results demonstrated a marked dependence on the timescale of the temperature profile of the mean square
displacement. Several dynamical transitions were observed
in the slower dynamics. Comparison with the temperature
profile of the activity of the enzyme in the same solvent
reveals dynamical transitions having no effect on GDH
function. Representing the first assessment of the global
dynamics of an active enzyme measured under similar
conditions over a range of time-scales, these studies suggest
that anharmonic, picosecond motions are not required at all
temperatures for the enzyme rate-limiting step. The authors
suggest that anharmonic fast motions are not necessarily
coupled to the much slower motions describing transitions
along the enzyme reaction coordinate. They caution,
however, that the neutron technique reveals average
dynamics, and it is conceivable that functionally important
fast motions may occur locally in the protein at the active
site, but below noise levels.
Eisenmesser et al. (2002) used magnetic resonance
spectroscopy to analyze conformational exchange in the
reaction catalyzed by prolyl-peptidyl isomerase (Reaction:
cis-X–Pro Isomer # trans-X–Pro Isomer), also known as
cyclophyllin A:
O
R1
H
N C
H
H
N
O
H
N
N
R2
C
H H
R1
O
R2
O
peptidyl-trans-proline
peptidyl-trans-proline
The simplest catalytic cycle consistent with known
catalytic properties is shown in Scheme 1.11, with three
microscopic reaction steps:
E
kcis,on
ktrans,on
ktrans,off
E-Prolinetrans
kcis,off
kc-to-t,cat
kt-to-c,cat
E-Prolinecis
Scheme 1.11
where Ktrans,D ¼ ktrans,off/ktrans,on and Kcis,D ¼ kcis,off/kcis,on.
Eisenmesser et al. (2002) conducted 15N spin relaxation
experiments in the absence and presence of the substrate
N-Succinyl-L-Ala-L-Phe-L-Pro-4-NA. Chemical-shift mapping
with 15N yields a single resonance (or a single peak in a twodimensional NMR spectrum) for each amide bond. By
changing the relaxation delay time, they determined the
transverse relaxation rate constant R2, which obeys the
relation: R2 ¼ R20 þ Rex. The latter represents the exchange
36
contribution to R2 and provides information about the
relevant motions that occur on the microsecond-to-millisecond time-scale. To separate the effects of binding from
cis-trans isomerization, the authors characterized substrate
concentration-dependent changes in R2. The relative
contributions to R2 from exchange due to binding and cistrans isomerization exhibited different dependencies on
substrate concentration. For most residues, Rex z pApBdv2/
kex where pA and pB are the fractional populations of free
enzyme and the bound states, dv is the chemical shift
difference between E and SE$Si, and kex is the exchange
rate. As substrate concentration is increased, Rex therefore
rises and then falls; maximal chemical exchange occurs at
intermediate substrate concentrations, where Efree z
(E$Scis þ E$Strans). The observed rate behavior fits with the
presence of significant concentrations of the three protein
forms (i.e., E, E$Scis, and E$Strans). When plotted as the 15N
R2 relaxation rate constant versus residue number (Fig. 1.8),
it became clear that certain regions in the enzyme exhibited
changes in R2 due to steady-state catalytic turnover. By
nonlinear regression analysis of the exchange contribution
to R2 for those residues sensing substrate binding and
catalysis, the authors obtained Km values ranging from 0.95
to 1.2 mM, and koff values of 10,700 to 14,800 s1.
Based on other quantitative estimates of the rate
constants for the protein’s structural dynamics, the
authors reached the important conclusion that areas
around residues 55, 82, 101–103, and 109 play a role in
substrate binding at or near the diffusion limit. After the
substrate is bound, the enzyme catalyzes a 180 -rotation
of the prolyl peptide bond, and the substrate tail on the
C-terminal side with respect to the prolyl residue is
viewed as swinging around to make contact with the
enzyme near residues 98 and 99. Meanwhile, the substrate’s N-terminal tail stays fixed, allowing the E$S
complex to remain intact, despite substantial rearrangements during the cis-trans isomerization at a rate of
9,000 s1. Most notably, motions of the substrate and the
enzyme coincide, and the catalytic Arg-55 also moves
with the same rate constant.
The beauty of this investigation on cyclophyllin A
catalysis is that Eisenmesser et al. (2002) succeeded in
identifying those regions of the enzyme whose dynamics
match the essential enzyme kinetics of catalysis. They also
mapped the microsecond time-scale dynamics to specific
regions of the cis-trans isomerase. Despite the fact that
additional analysis is needed to define the motions during
the actual catalytic event, their systematic approach defined
dynamic ‘‘hot spots’’ during catalysis and revealed that the
time-scales for protein dynamics coincide with those for
substrate turnover. Finally, Bosco, Eisenmesser and Kern
(2002) also described CypA’s catalytic action on Pro-90 in
the HIV capsid protein. Their work is the first documented
case of catalyzed cis-trans isomerization on a prolyl residue
within a natively folded protein substrate.
Enzyme Kinetics
FIGURE 1.8 Residues in cyclophyllin A exhibiting microsecond timescale dynamics during catalysis. Structures of the enzyme-bound cis and
trans conformations of the substrate N-Succinyl-Ala-Phe-Pro-4-NA
(green) bound to the enzyme (including expanded views shown at right),
based on the X-ray structure of CypA complexed to the cis form of
N-Succinyl-Ala-Phe-Pro-4-NA (1RMH) (Zhau and Ke, 1996). CypA residues with chemical exchange in both the presence and absence of substrate
are color-coded in blue (namely Phe-67, Gln-71, Gly-74, Ser-77, and Ser100). Residues in red exhibit chemical exchange only during turnover
(Arg-55, Lys-82, Leu-98, Ser-99, Ala-101, Gln-102, Ala-103, and Gly109). Residues shown in magenta (Thr-68 and Gly-72) exhibit chemical
exchange in the absence of the substrate but increase in its presence.
CypA catalyzes prolyl isomerization by rotating the C-terminal part of
the prolyl peptide bond by 180 to produce the trans conformation of
the substrate. In this model, the observed exchange dynamics of residues
in strand-5 of the enzyme can be explained. Reproduced from Eisenmesser
et al. (2002) with the permission of the authors and the American Association for the Advancement of Science.
Earlier two-dimensional heteronuclear (1H–15N) nuclear
magnetic relaxation studies suggested that the dihydrofolate
reductase$dihydrofolate complex exhibits a diverse range of
backbone fluctuations on the psec-to-nsec time-scale
(Epstein, Benkovic and Wright, 1995). To assess whether
these dynamical features influence Michaelis complex
formation, Miller and Benkovic (1998) used mutagenesis
and kinetic measurements to assess the role of the strictly
conserved residue Gly-121, which displays large-amplitude
backbone motions on the nanosecond time-scale. Deletion
of Gly-121 dramatically reduces the hydride transfer rate by
550 times; there is also a 20-times decrease in NADPH
cofactor binding affinity and a 7-fold decrease for NADPþ
relative to wild type. Insertion mutations significantly
decreased both substrate and cofactor binding. Their results
suggest that distant residues, such as Gly-121 in DHFR,
Chapter j 1 An Introduction to Enzyme Science
may influence the formation of liganded complexes as well
as the proper orientation of substrate and cofactor during the
catalytic cycle.
Finally, it is also worthwhile to ponder a related question:
Why are enzymes so large? Aside from the structural
complexity of allosteric enzymes, the most common answer
is that most enzymes are made up of domains and motifs,
the binding properties of which have been honed through
Natural Selection. In numerous lectures on chemical and
enzyme catalysis, the late Daniel Koshland was fond of
comparing a hydroxide ion to a hand-drill and an enzyme to
a milling machine. His point was that enzyme catalysis
almost certainly requires highly precise interactions of an
enzyme with its substrate(s). In the context of transitionstate stabilization, it is reasonable to anticipate that a strong
and exact fit of enzyme and substrate within the (E$X)z
transition-state complex is critically important. Similarly,
binding energy is an essential ingredient for ground-state
destabilization. From the perspective of enzymes as forceactuated catalytic devices, however, the oil-like properties
of the hydrophobic cores of globular enzymes may absorb,
redirect, and align forces imparted by thermal energy with
respect to the trajectory of E$S along the reaction coordinate. In this respect, domains may also focus these forces at
critical stages within the catalytic cycle, much like lattice
dislocations are thought to facilitate heterogeneous catalysis
on metal surfaces. While the breaking of discrete chemical
bonds occurs on the picosecond time-scale, protein
conformation changes occur on the same nanosecond-tomicrosecond time-scale, as is observed for enzyme catalysis. Any force F exerted over a distance Dx along the
reaction coordinate should have the effect of reducing the
zero-force DEact,0 value to the effective activation energy
DEact,effective, such that DEact,effective ¼ DEact,0 – FDx. A
large protein may even have the effect of increasing the
magnitude of Dx, thereby further reducing the effective
activation energy.
1.6.2 We Need New Approaches for
Determining the Channels Allowing Energy
Flow During Enzyme Catalysis
A related outstanding problem in enzyme science concerns
the if’s, where’s, when’s, and how’s of energy flow within
enzyme molecules during catalysis. For nearly a century, the
main approach of enzyme chemists has been the determination of the enzyme-catalyzed transformations of substrates
to intermediates and thence to products, without due
consideration of how enzymes might manipulate the flow of
energy to achieve their enormous catalytic rate enhancements. The origin of that energy and its detailed path(s)
within an enzyme molecule could, in principle, explain why
Nature relies on such a small number of proteins for catalysis. Current estimates put the number of different enzymes
37
at around 20,000 to 30,000 for all species, not counting many
billions of largely inconsequential, but naturally occurring
amino acid substitutions; however great the number of such
naturally occurring enzymes, the combinatorial variability of
polypeptides, with say 400 residues, would be an astonishingly great (~20400). Present day ideas about critical enzyme
residues and the focused flow of energy within proteins are
best characterized for redox proteins like the cytochromes,
rhodopsin, green fluorescent protein, as well as photosynthetic reaction centers. Even then, the actual energy-flow
pathways are at best sketchy.
As reviewed by Leitner (2008), energy flow within
a protein may be treated as a percolation process involving
a network of sites, some resulting in fast transport when
distant points are directly connected by energy-flow channels, with others exhibiting slow transport along numerous
pathways that most often reach dead ends. This connection
can be made more precise by comparing statistically energy
flow in proteins with flow related to the nature and density
of a protein’s vibrational states. Energy transfer can occur as
molecular vibrations or by dipole–dipole interactions in
photoexcited states. The former, which is limited by the
speed of sound and is most frequently carried by the relatively low-frequency modes of a protein, occurs on the order
of 10 Å/psec1. With a mean free path on the order of 1 Å,
the shortest time over which diffusion can be observed is
around 0.1 picoseconds. For proteins consisting of 100
residues, energy diffuses from the interior to the surface in
a few picoseconds, so vibrational energy flow in proteins
exhibits anomalous subdiffusion, with times of approximately 0.1 picoseconds. Although the nature of fluorescence
resonance energy transfer (FRET) will be described in
Section 4.5.6, it is sufficient here to say that the efficiency of
FRET depends on the relative orientation of the donor and
acceptor moieties as well as the distance between them,
with the latter imposing an inverse sixth power dependence
on that distance. When the acceptor is photo-emissive,
a photon will be emitted after a red-shift (i.e., the donor will
absorb shorter wavelength light than that emitted by the
acceptor), and the timescale will depend mainly on the
fluorescence lifetime of the acceptor. When the acceptor is
a quencher, one must entertain the possibility that the
resulting thermal energy of the excited-state acceptor may
conceivably be channeled into discrete vibrational modes
and/or conformational changes.
Little solid information exists concerning the internal
transmission of energy within enzymes, and even less is
known for enzymes during catalysis. The extreme celerity
of enzymic catalysis imposes both technical limitations on
the detection and quantification of transient changes in
enzyme structure as well as substantial uncertainties
regarding the positions, motions, and momenta of critical
catalytic residues. Even a fraction of an Ångström in the
position of a catalytic functional group could easily spell the
difference between a poor and highly efficient catalyst.
38
1.6.3 We Need Additional Probes
of Enzyme Catalysis
To define the mechanisms of energase-type mechanochemical reactions, one must learn how the DGhydrolysis (or
DGelectron-transport) drives protein transitions between noncovalent substrate- and product-like interaction states.
Although modern protein science seeks to understand how
conformational energy is generated, stored, and managed,
there are as yet no rules for predicting likely reaction
intermediates and transition states in energase catalysis.
From this perspective, the task of elucidating energase
mechanisms represents a monumental challenge for enzymologists and structural biologists alike. Although fluorescence and Förster resonance energy transfer are powerful
tools for detecting and quantifying noncovalent interactions
and conformational transitions, Ångström-scale resolution
is needed to unambiguously define structural alterations that
attend enzyme catalysis. Enzyme science will therefore
benefit enormously by the development of additional
spectroscopic and crystallographic tools capable of
discerning the small structural changes occurring in the
enzyme during catalysis. When coupled with appropriate
computer-based modeling of enzyme interactions with
substrates and inhibitors, a fuller picture of catalysis should
emerge.
The promise of time-resolved X-ray crystallography
must not be underestimated. Of particular note is the Laue
diffraction method, which uses polychromatic X-rays
(typically l < 2.0 Å) to collect sufficient structural data to
compute a series of images on a short time-scale (Moffat,
2001). Although Laue diffraction and computational
molecular dynamics (MD) were developed as independent
ways to visualize and assess transient structural states,
their combined use may allow mutual refinement of
computational MD simulations of Michaelis complexes
and difference Fourier electron density maps obtained in
Laue experiments. Because a realistic molecular dynamics
study of a 50-kDa protein requires one to determine the
positions of ~10,000 atoms, every 1015 seconds, largescale MD simulations necessarily create huge data sets.
The technique known as Principal Component Analysis is
a mathematical tool for detecting correlations in large data
sets. By expressing a molecular dynamics trajectory as
a linear combination of principal components, the background atomic fluctuations (i.e., thermal noise) are eliminated, affording a better view of the protein’s collective
motions (Balsera et al., 1996; Hayward, Kitaom and Go,
1994; Mongan, 2004). When combined with appropriate
physical models for protein motion, PCA can help one to
detect genuine conformational changes. For example,
mutual use of MD and crystallographic refinement allowed
Stoddard, Dean and Bash (1996) to assign a number of
additional contacts and features for hydride transfer by
isocitrate dehydrogenase. They reported that unrestrained
Enzyme Kinetics
independent MD simulations provide a very useful crossvalidation method for highly mobile regions that exhibit
poorly defined experimental density. Likewise, information from Laue difference maps provides information
about substrate conformation and interactions that greatly
facilitate MD simulations.
In a truly formidable undertaking, Schmidt et al.
(2003) successfully determined the number of authentic
late-stage photo-cycle intermediates of PYP, the 14-kDa
photoactive yellow protein from the purple eubacterium
Ectothiorhodospira halophila. PYP possesses a 4hydroxycinnamic acid chromophore linked as a thiolester
to Cys-69. Its 446-nm lmax matches the action spectrum
for negative phototaxis, suggesting that PYP is the
primary cytoplasmic blue-light photoreceptor for this
process. Schmidt et al. (2003) used laser light absorption
to trigger the series of room temperature chemical reactions in PYP crystals, and they then employed the Laue
diffraction technique (see 10.6.1: Flash Photolysis) to
determine atomic structures of PYP after a laser-to-X-ray
interval of 5 ms, 9 ms, 20 ms, 51 ms, 125 ms, 250 ms, 500
ms, 850 ms, 1 ms, 2 ms, 7 ms, 15 ms, 30 ms, or 100 ms.
They applied singular value decomposition (SVD) to the
series of experimental, time-dependent difference maps.
This approach allowed them to evaluate rival chemical
kinetic mechanisms and to arrive at a self-consistent
mechanism through their analysis of a set of timedependent difference electron density maps spanning the
time range from 5 ms to 100 ms. Successful fit of
exponentials to right singular vectors derived from
a singular value decomposition of the difference maps
demonstrates that a chemical kinetic mechanism holds,
and that structurally distinct intermediates exist.
Schmidt et al. (2003) identified two time-independent
difference maps, from which they refined the structures of
the corresponding intermediates, thereby demonstrating
how structures associated with intermediate states can be
extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints
allowed them to exclude one kinetic mechanism proposed
for the photocycle but retain other plausible candidate
kinetic mechanisms. Thus, despite the fact that some might
justifiably quarrel with this author’s views as to whether
a 446-nm photon is truly a substrate or whether the PYP
photocycle is catalytic, the approach taken by Schmidt et al.
(2003) represents a bench-mark in pioneering efforts to
analyze the time-evolution of an enzyme’s structure during
catalysis.
1.6.4 We Need to Learn How Proteins Fold
and How to Manipulate Protein Stability
Determining how proteins fold is also an enterprise of
central significance to enzymology, both with respect to
how unfolded polypeptide chains self-organize to form
Chapter j 1 An Introduction to Enzyme Science
active catalysts and how molecular chaperonins facilitate
such processes. The challenge is to conceive of and execute
experiments that reveal the time-evolution of evanescent
short-, medium- and long-range structures adopted by
a protein during its folding and to develop adequate theories
and simulation algorithms that capture essential features of
the folding process. Given the fact that folding can now be
viewed as the consequence of a massive, parallel ‘‘diffusional’’ search of n-dimensional conformational space, the
idea that discrete intermediates accumulate would imply
that there are kinetically significant bottlenecks in the
folding process. In their remarkable paper, Laurents and
Baldwin (1998) discuss how the image of the transition state
has changed from a unique species (with a strained
configuration and a correspondingly high free energy) to
a more ordinary folding intermediate reflecting a balance
between limited conformational entropy and stabilizing
contact places. As they explain, evidence for a broad transition barrier comes from studies showing that mutations
can change the position of the barrier. Controversy remains
as to whether populated folding intermediates (i.e., those at
detectable concentrations) are productive ‘‘on-pathway’’
intermediates or ‘‘dead-end’’ traps. Another confounding
issue concerns the generalizability of folding rules discovered to govern a particular protein. While these topics lie
well beyond the scope of this monograph, readers should
consult Dobson and Fersht (1995), Fersht (1998), and
Richards et al. (2000).
1.6.5 We Need to Develop a Deeper
Understanding of Substrate Specificity
Understanding enzyme specificity remains an enormous
unfulfilled challenge for structural biologists and enzyme
chemists alike. Learning the rules governing substrate
specificity is essential in efforts to craft new metabolic
pathways – a task of ever-greater significance in the design
of microorganisms tailored to produce new plastics,
renewable fuels, and novel therapeutics. Enzymes of 50kDa molecular mass have a molecular volume of ~100 nm3,
and their active sites are located in ~1-nm3 clefts and
crevices. In a sense, the complex, self-adaptive chemical
process that we call Life is only possible because each of
these 1-nm3 clefts and crevices exhibits a limited repertoire
of bio-specific interactions. Most active sites bind substrates
and/or coenzyme with a combined molecular weight of
800–1,200 Daltons. The challenge of understanding enzyme
specificity not only speaks to the need for high-resolution
enzyme structures but also for kinetic data indicating how
subtle changes in enzyme structure determine interactions
with substrates and inhibitors. If generalizable rules for
enzyme specificity can be discovered, it should be possible
to rebuild and/or remodel active sites to accommodate new
substances as substrates.
39
Directed evolution of novel,9 catalytically proficient
enzymes is quickly emerging as a powerful new theme in
enzyme science. Biochemists are seeking to modify
substrate recognition, to eliminate side-reactions, to form
specific products, and to increase catalytic turnover rates.
Such efforts have traditionally been limited by the selection
(or screening) method. In vivo selections are usually
restricted to identifying properties affecting the viability of
the organism, and full exploitation of these approaches is
often compromised by the complex nature of a living cell’s
intracellular environment and the need to transform that
cell’s gene-library. Typically, 103–105 clone libraries are
screened in a plate assay using a fluorogenic or chromogenic substrate to identify a few colonies of interest. To alter
enzyme enantiomeric specificity for eventual use in asymmetric organic synthesis, Reetz et al. (1997) proposed
a general approach that does not require any knowledge of
the structure or the mechanism of the enzyme, namely in
vitro evolution using a combination of random gene mutagenesis by error-prone PCR (Leung, Chen and Goeddel,
1989) and subsequent expression and high-throughput
screening. To achieve error-prone Polymerase Chain
Reaction (or epPCR), the reaction conditions are varied
empirically to reduce Taq polymerase fidelity during DNA
amplification, thereby causing base substitutions resulting
in one, two, three, or even more amino acid substitutions in
the encoded protein. Reetz (2004) discussed the scope and
limitations of directed mutagenesis approaches, including
the prospect of obtaining stereoselective hybrid catalysts
composed of robust protein hosts in which transition metal
centers have been implanted. Some efforts have focused on
using in vitro compartmentalization (IVC), an ingenious
approach wherein a reaction assay solution can be
9
When biochemists most often use the word ‘‘novel’’ to describe
a substance, reaction, enzyme, etc., they are indicating that, to the
best of their knowledge, no such biochemical substance or reaction
has been previously reported. From a biological perspective, such
substances, reactions, enzymes, etc., are not new inasmuch as they
have presumably been essential components for a long time. Given
the introduction of manmade chemical substances into the
environment for nearly two centuries, however, there is an increased
likelihood for inadvertent evolution to give rise to a truly novel
enzymatic activity. A case in point is bacterial phosphotriesterase,
a microbial enzyme that catalyzes the hydrolysis of a broad range of
phosphotriester substrates, including the neurotoxic cholinesterase
inhibitors paraoxon (diethyl p-nitrophenyl-phosphate) and parathion
(diethyl p-nitrophenyl-thiophosphate). As discussed by Shim, Hong
and Rauschel (1998), the rarity of naturally occurring phosphotriester
substrates suggests that phosphotriesterase catalysis may be truly
novel and that no such activity occurred prior to the introduction of
these agents into the environment. Biochemists are also interested in
directed enzyme evolution as a way to create new metabolic
pathways or to improve chemical syntheses. Efforts to modify the
chemical and/or kinetic properties of enzymes or to make catalysts
from previously non-catalytic proteins and nucleic acids also raise the
likelihood for observing truly novel enzymatic activities.
Enzyme Kinetics
40
partitioned into microscopic compartments, each of only ~5
fL, by forming water-in-oil emulsions. In this way, a 50-mL
reaction volume can be dispersed into 1010 physically isolated, aqueous compartments, allowing for the selection of
many genes and making the system highly sensitive and
economical. Tawfik and Griffiths (1998) and Lee, Tawfik
and Griffiths (2002) demonstrated the feasibility of using
IVC to select DNA methyltransferases. Likewise, Levy,
Griswold and Ellington (2005) used a compartmentalized in
vitro selection method to directly select for ligase ribozymes
that are capable of acting on and turning over separable
oligonucleotide substrates. Starting from a degenerate pool,
they selected a trans-acting variant of the Bartel class I
ligase that statistically was likely to be the only active
variant in the starting pool, and isolation of this sequence
from the population suggests that this selection method is
extremely robust at selecting optimal ribozymes.
As a concrete example of a directed evolution experiment,
consider the work of Griffiths and Tawflik (2003) on the
selection of a high-kcat phosphotriesterase with turnover rates
>105 s1, some 63 higher the wild-type enzyme. Mutant
enzymes were selected from a library of 3.4 107 mutated
phosphotriesterase genes using the ingenious strategy of
linking genotype and phenotype by means of in vitro
compartmentalization (IVC) in water-in-oil emulsions. First,
microbeads, each displaying a single gene and multiple copies
of the encoded protein, were formed by compartmentalized in
vitro translation. To select for catalytic properties, the
microbeads were re-emulsified in a reaction buffer containing
a soluble substrate, and the product and any unreacted
substrate were coupled to the beads when the reaction rate
assay was complete. Product-coated beads, displaying active
enzymes and the genes that encode them, were detected with
anti-product antibodies and selected using flow cytometry.
With this completely in vitro approach, Griffiths and Tawflik
(2003) were able to select for substrate recognition, product
formation, rate acceleration and turnover.
Kim et al. (2001) simultaneously incorporated and
adjusted functional elements within an existing enzyme by
inserting, deleting, and substituting several active-site loops,
followed by fine-tuning of catalytic properties by means of
site-directed point mutation. They successfully introduced
b-lactamase activity into the ab/ba-metallohydrolase scaffold of glyoxalase II, and the re-engineered enzyme lost its
original activity and gained the ability to catalyze the
hydrolysis of cefotaxime with a (kcat/Km)app value of 1.8 102 M1 s1. While this specificity constant value is rather
low, Escherichia coli containing the redesigned enzyme
exhibited 100 greater resistance to cefotaxime. The
potential for extending these efforts by combining sitedirected-mutagenesis and chemical modification to improve
the specificity of enzymes, especially those used by synthetic
organic chemists, should not be underestimated (Jones and
Desantis, 1998) (see also Section 2.3: Active Site
Diversification).
An intriguing case of substrate specificity is the
carboxylase/oxygenase, the
CO2-fixing enzyme that exhibits relatively slow catalysis
attributed to the need to discriminate between its substrates
CO2 and O2. Tcherkez, Farquhar and Andrews (2006)
argued that these characteristics arise from difficulty in
specific binding of the structurally featureless CO2 molecule, forcing substrate specificity for CO2 versus O2 to be
determined later (i.e., in the transition state). They suggest
that natural selection for greater CO2/O2 discrimination, in
response to reducing atmospheric [CO2]/[O2] concentration
ratios, resulted in a transition state for CO2 addition that
resembles a carboxylate group. This adaptation maximizes
structural differences between transition states for
carboxylation and oxygenation. However, the resulting
increased similarity between the structure of the carboxylation transition state and its carboxyketone product
exposes the carboxyketone to the strong binding needed to
stabilize the transition state, causing the carboxyketone to
bind so tightly that its cleavage to products is slowed.
Tcherkez, Farquhar and Andrews (2006) suggested that
such apparent compromises in catalytic efficiency for the
sake of specificity represent a new type of evolutionarily
perfected enzyme.
Substrate specificity also reinforces the idea that
enzymes are ideally suited for the synthesis and/or derivitization of drugs. Consider, for example, the studies of
Khmelnitsky et al. (1997) focusing on the synthesis of
water-soluble forms of paclitaxel (taxol), the potent anticancer drug that binds selectively to assembled microtubules. Scheme 1.12 shows that in the absence of any
selective functional group protection, these investigators
identified a two-step enzymatic process for selective acylation and deacylation.
There are two potentially reactive hydroxyl groups
(marked in red), but thermolysin selectively transfers the
adipoyl moiety to only one, thereby preventing loss of
biological activity by modification of the taxane ring.
Likewise, only one of the two ester-linkages (marked in
blue) is cleaved by the fungal lipase. Notice that both
reactions occur in polar organic solvents.
There is also good reason to believe that biochemists
have not as yet identified all of the physiologically
significant ligands – even for those enzymes already
thought to be well characterized. The search for enzyme
regulatory molecules is often hit-or-miss, as evidenced by
the serendipitous discovery of the pivotally important
allosteric effector Fructose-2, 6-P2 as well as the recent
unanticipated development of synthetic glucokinase activators. In fact, we have no way to reckon just how many
central pathway activators and inhibitors remain to be
discovered. Moreover, although most enzymes are first
discovered and isolated through the use of a well-defined
activity assay, one can never be absolutely certain that
a particular substrate is the physiologic substrate or that
D-ribulose-1,5-bisphosphate
Chapter j 1 An Introduction to Enzyme Science
H3 C
41
O
Ph
OH
O
H3C
O
NH
O
CH 3
CH 3
Ph
O
OH
HO
O
CH 3
O
Ph
Divinyl Adipate
in
tert-Amyl Alcohol
Thermolysin
(Salt-Activated)
H3 C
O
Ph
OH
O
H3 C
O
NH
O
CH 3
CH 3
Ph
O
O
C O
OH
CH 2 =CH—O-C(=O)—(CH2)3—CH 2
O
CH 3
Ph
Acetonitrile
(solvent)
O
Lipase
Candida antarctica
H3C
O
Ph
OH
O
H3 C
O
NH
O
CH 3
CH 3
Ph
OH
O
O
C O
HOOC—(CH 2)3—CH 2
O
CH 3
Ph
O
Scheme 1.12
other substrates are also metabolized. Many enzymes are
selective in their action toward substrates and are only
rarely exhibit absolute specificity. Nowhere is this statement truer than in the identification of the primary phosphoryl-acceptor substrate for the numerous signaltransducing protein kinases. An added issue is the
phenomenon of ‘‘catalytic promiscuity’’ (see Section
2.3.2), wherein a single enzyme operates by more than one
catalytic mechanism, giving rise to multiple enzymatic
activities. Catalytic promiscuity increases the likelihood
that we have unknowingly failed to identify many physiologically important reactions.
Such concerns point the need for a far more comprehensive
X-ray and NMR investigation of many, many more enzymes
to define the structures of their active sites and regulatory sites
at atomic resolution. Consider the fact that the Protein Data
Bank (PDB) presently lists some 56,000 structures, with
nearly one-fourth of human origin. Some 49,000 structures
were established by X-ray techniques, with 7,000 determined
by NMR and fewer than 200 by EM. Also listed in the PDB
are ~2,100 nucleic acid structures, with ~1,200 from X-ray
analysis, ~900 from NMR, and <20 from EM. For the nearly
2,500 structures for protein-nucleic acid complexes, ~2,300
were determined by X-ray, ~150 by NMR, and <65 by EM.
While the tally of 56,000 documents the impressive pace of
acquiring protein structures over the past half century, it gives
a somewhat distorted view of how much we already know,
simply because the ligand-free and -bound structures and
mutant forms of certain proteins have been so intensively
investigated that these proteins are disproportionately represented in the PDB. Various hemoglobins, for example,
account for ~1.6% of all PDB structures. Among the intensively studied enzymes are: lysozyme (2%), angiotensinogen-converting enzyme (~1.5%), RNase (~1.4%), the
ribosome (~1.4%), trypsin (1.3%), chymotrypsin (~1%),
actin (1%), carbonic anhydrase (0.6%), adenylate kinase
(~0.5%), and myosin (~0.4%). Collectively, the proteins listed above represent one-eighth of all curated structures in the
PDB! To fathom the degree to which the overall tally grossly
under-represents the proteome, one need only consider that
human and mouse genomes each contain >20,000 proteinencoding genes, with Drosophila at ~13,000, C. elegans at
~17,000, Arabidopsis at ~28,000, rice at ~38,000, S. cerevisiae at ~6,000, and E. coli at ~5,000. In all, more than
500,000 proteins would be needed to represent the proteomes
of the 100 most frequently studied organisms and viruses.
Even after allowing for the 10–15% that are fibrous and/or
intrinsically disordered, upwards of 3–5 million different
protein structures would be required to fully represent the
ligand-free and -bound states for the remaining globular
proteins. An effort exclusively directed toward defining the
structures of all human proteins would itself swell the current
PDB holdings by a factor of 5–10. Obviously, such a massive
undertaking is presently infeasible and would require development of high-throughput robotic methods for efficiently
expressing, purifying, crystallizing, and then structurally
analyzing such a vast array of protein structures.
To reveal telltale structural features underlying molecular recognition and substrate specificity, one need not
possess an atomic-level structure for all enzymes within
a proteome. One may only need the structures of as few as
five to ten thousand more enzymes with numerous representatives from each reaction types found in the Enzyme
Commission’s classification. Moreover, wider application
of molecular docking with a suitably robust reference
library consisting of all known low-molecular-weight
metabolites would develop criteria for reliably predicting
the most substrate specificity as well as the probable catalytic mechanism for those enzymes whose activities have
Enzyme Kinetics
42
yet to be established experimentally. Computational
approaches are also required to provide a means for efficiently re-surveying enzyme surfaces, again at atomic
resolution, to find previously undiscovered crevices that
serve as activator and inhibitor sites. Such work may also
help us to understand how so many different proteins
manage to co-exist within crowded compartments with
engaging in nonspecific aggregation.
Although highly automated robotic acquisition of
enzyme structures may provide us with a catalogue of highresolution structural data, the value of such a treasure trove
of structural data would be underwhelming in the absence of
commensurate advances in high-throughput biochemical
characterization. What ultimately drives discovery science
is the sense of intrigue and opportunity that researchers
experience when they ponder the properties and complexity
of an unsolved scientific problem. Without commensurate
growth in hypothesis-based, experimental enzymology, we
would soon find, as put so well by Tennyson, that
‘‘knowledge comes, but wisdom lingers.’’ Structural and
functional characterization of the entire human proteome
would allow us to comprehend the full spectrum of ligand
binding interactions underlying enzyme catalysis and
control as well as to manage disease-causing enzyme
mutations through the design of new drugs and/or therapeutic interventions.
To date, most molecular structure analyses stem from an
interest in a particular enzyme or its intriguing biochemical
properties; even so, there is good reason to believe that we
have not succeeded in identifying likely physiologically
significant alternative substrates or all of the allosteric
activators and inhibitors. There is thus an emerging
recognition of the need for a more comprehensive investigation of numerous active-site structures at atomic resolution by X-ray and neutron crystallography. Such
a coordinated effort, which would focus on perhaps as few
as several thousand more enzymes representing every
Enzyme Commission reaction type, is likely to reveal
telltale structural features that underlie substrate specificity.
Moreover, wider application of molecular docking with
a suitably robust reference library consisting of all known
low-molecular-weight metabolites (i.e., MW < 1–3 kDa)
would develop criteria for reliably predicting the most
substrate specificity as well as the probable catalytic
mechanism for those enzymes whose activities have yet to
be established experimentally. Efforts to perfect highthroughput computational approaches are also required to
provide a means for re-surveying all enzyme surfaces, again
at atomic resolution, thereby fostering the development of
new ways to predict previously undiscovered activator and
inhibitor sites. Such efforts would fulfill a longstanding
need to comprehend the fuller spectrum of ligand binding
interactions responsible for cell, tissue, organ, and interorgan regulatory mechanisms. These same approaches can
be extended to the systematic investigation of naturally
occurring, disease-causing enzyme mutations, perhaps even
facilitating the design of custom-tailored therapeutic
interventions.
Finally, by redesigning enzyme active sites to accommodate novel substrates, we face the welcome prospect that
therapeutic enzymes may soon be re-fashioned in ways
allowing them to modify and/or detoxify natural and
manmade toxins. Given the many millions of synthetic
organic chemicals that have been prepared for commercial
and research purposes, the ability to re-jigger enzyme active
sites to catalyze novel reactions would increase the remedial potential during failures in chemical containment,
especially if existing highly abundant enzymes can be
altered for such purposes. We may likewise anticipate the
use of these synthetic enzymes in the conversion of prodrugs (see Section 8.12.5) into their therapeutically active
forms.
1.6.6 We Need to Develop the Ability to
Design Entirely New Biological Catalysts
Given the trend toward minimizing the environmental
impact of chemical industries, greater emphasis must be
placed on designing enzymes with new catalytic function.
Learning precisely how substrates approach and dock
within enzyme active sites should permit us to remodel
active sites to create new catalysts.
Shown in Fig. 1.9 is the enlightening and efficient multistep strategy developed by Jiang et al. (2008) for the rational
design of new enzymes, with their study focusing on the
catalysis of retro-aldol reaction (Scheme 1.13).
H3C
C
CH2
O
O
CH
CH3
H3C
Enzyme
H3C
C
O
CH3
O
HC
CH3
O
Scheme 1.13
In the first step of their computational enzyme design
effort, Jiang et al. (2008) defined potential catalytic mechanisms for a retro-aldol-type reaction. Recall that this
reaction proceeds in distinct stages (Scheme 1.14), each
involving acid/base catalysis by either amino acid side
chains or water molecules (see also Fig. 2.27 describing
aldolase catalysis).
Chapter j 1 An Introduction to Enzyme Science
H-bond
O
OH
43
H-bond
:B
OH
:B
:B
LYS
OH
NH
H2O
LYS
HN
NH2
O
O
LYS
H-bond
O
LYS
NH2
OH
:B
H-bond
:B
H
OH
OH
+
O
O
:B
H2O
+
HN
H2N
LYS
LYS
HN
O
LYS
Scheme 1.14
Nucleophilic attack of an enzyme lysine on the substrate’s ketone group forms a carbinolamine intermediate,
which upon eliminating water forms the imine/iminium
species. Carbon–carbon bond cleavage is then triggered by
the deprotonation of the b-alcohol, with the iminium
intermediate acting as an electron sink. Finally, the enamine
tautomerizes to an imine, which is then hydrolyzed to
release the covalently bound product and free the enzyme
for another round of catalysis. Each elementary reaction in
such a multi-stage mechanism has its own transition state,
which must be stabilized by the enzyme.
In the second step of the design process, Jiang et al.
(2008) identified known protein scaffolds that might
accommodate the designed TS ensemble described above.
To account for the multi-step reaction pathway, they
designed a composite structure of acid/base groups that is
simultaneously compatible with multiple transition states
and anticipated reaction intermediates. In this effort, they
generated design models using the four catalytic motifs
shown schematically in Fig. 1.10, which employ different
constellations of catalytic residues to facilitate carbinolamine formation and water elimination, carbon–carbon
bond cleavage, and release of bound product. The authors
emphasize that it is essential to consider a very large set of
active-site possibilities, simply because the probability of
accurately reconstructing a given three-dimensional active
site in an input protein scaffold is extremely small. They
generated such a set by simultaneously varying: (i) the
internal degrees of freedom of the composite TS; (ii) the
orientation of the catalytic side chains with respect to
the composite TS, within ranges that are consistent with
catalysis; and (iii) the conformations of the catalytic side
chains. This combinatorial matching resulted in a total of
181,555 distinct solutions for the placement of the
composite TS and the surrounding catalytic residues.
The Rosetta Match algorithm rapidly eliminated most
active-site possibilities in a given scaffold that are likely
to have unfavorable catalytic geometry or to give rise to
significant steric clashes.
After optimization of the composite TS rigid body
orientation and the identities and conformations of the
surrounding residues, a total of 72 designs with 8–20
amino acid identity changes in 10 different scaffolds were
selected for experimental characterization based on the
predicted TS binding energy, the extent of satisfaction of
the catalytic geometry, the packing around the active
lysine, and the consistency of side-chain conformation
after side-chain repacking in the presence and absence of
the TS model. cDNA’s encoding each design were constructed and the proteins were expressed and purified from
Escherichia coli, yielding soluble purified protein for 70
of 72 designs.
Retro-aldolase activity was monitored via a fluorescence-based assay of product formation for each of the
designs. Their initial 12 designs used Motif I (Fig. 1.11B),
which involves a charged side-chain (Lys-Asp-Lys)-mediated proton transfer scheme resembling that for D-2deoxyribose-5-phosphate aldolase. Of these designs, two
showed slow enaminone formation with 2,4-pentandione
(17), which is indicative of a nucleophilic lysine, but none
displayed retro-aldolase activity. Ten designs were made
based on Motif II, which is much simpler and involves
a single imine-forming lysine in a hydrophobic pocket,
similar to aldolase catalytic antibodies. Of these designs,
one formed the enaminone, but none were catalytically
active. The third active site (Motif III) incorporates a HisAsp dyad as a general base to abstract a proton from the
b-alcohol; of the fourteen designs tested, ten exhibited
stable enaminone formation, and eight had detectable retroaldolase activity. In Motif IV, Jiang et al. (2008) experimented with the explicit modeling of a water molecule,
positioned via side-chain hydrogen bonding groups, which
shuttles between stabilizing the carbinolamine and
abstracting the proton from the hydroxyl. Of the thirty-six
Enzyme Kinetics
44
Compute TS for each step
with optimally placed
protein functional groups
Select library of
scaffold proteins
Combine to generate
composite active site
Identify pockets
Identify scaffold positions allowing construction of active site
Optimize composite TS and catalytic side-chain conformations
Design neighboring positions for high affinity TS binding
Optimize entire active site
Rank based on binding energy and catalytic geometry
Experimentally characterize top ranking designs
FIGURE 1.9 Computational design protocol for a multi-step enzymecatalyzed reaction. Step-1: Generate ensembles of models of each of the
key intermediates and transition states (TS) in the reaction pathway in the
context of a specific catalytic motif composed of protein functional groups.
Step-2: Superimpose these models, based on the protein functional group
positions, to create an initial composite active-site description. Step-3:
Generate large ensembles of distinct 3D realization of these composite
active sites by simultaneously varying the degrees of freedom of the
composite TS, the orientation of the catalytic side chains relative to the
composite TS, and the internal conformation of the catalytic side chains.
For each composite active site description, candidate catalytic sites are
generated in an input scaffold set by Rosetta Match software (Zanghellini
et al., 2006). Briefly, each rotamer of each catalytic side-chain is placed at
each position within each scaffold, and the ensuing position of the composite
TS is recorded in the hash. After filling out the hash table, which is linear in
the numbers of scaffold positions and catalytic rotamers, the table is
searched for TS positions (termed ‘‘matches’’) that are compatible with all
catalytic constraints; such positions are termed ‘‘matches.’’ Step-4: Optimize the rigid body orientation of the composite TS and the internal coordinates of the catalytic side chains for each match, reducing steric clashes
while maintaining the catalytic geometry within specified tolerances. The
remaining positions (not included in the minimal catalytic site description)
surrounding the docked composite TS model are redesigned to optimize TS
binding affinity by means of the standard Rosetta design methodology (Dantas et al., 2003; Meiler and Baker, 2006). The rigid body orientation of the
composite TS, the side chain torsion angles, and (in some cases) the backbone torsion angles in the active site are refined via quasi-Newton optimization. Step-5: Rank the resulting designs, based on the total binding energy to
the composite TS and the satisfaction of the specified catalytic geometry.
Step-6: Experimentally characterize the top-ranked designs. Figure and
legend reproduced with minor modification from Jiang et al. (2008) with
permission of the authors and the publisher.
designs tested, twenty formed the enaminone and twentythree (with eleven distinct positions for the catalytic lysine)
had significant retro-aldolase activity, with rate enhancements up to four orders of magnitude over the uncatalyzed
reaction.
To evaluate the accuracy of the design models, Jiang
et al. (2008) solved the structures of two of the designs by
X-ray crystallography (Fig. 1.11). The 2.2-Å resolution
structure (Panel D) showed that the designed catalytic
residues Lys159, His233, and Asp53 superimpose well on
the original design model, and the remainder of the active
site is nearly identical to the design. The 1.9 Å resolution
structure of the M48K variant of RA61 likewise reveals an
active site very close to that of the design model, with only
His46 and Trp178 in alternative rotamer conformations,
perhaps resulting from the absence of substrate in the crystal
structure (Panel E).
What is so appealing about the work of Jiang et al. (2008)
is that each proposed catalytic mechanism is treated as an
experimentally testable hypothesis through multiple independent design experiments. A candidate scaffold with its
pendant catalytic groups can first be tested in silico by
computer modeling protocols, then in vitro by kinetic
measurements, and finally in the crystal state by X-ray
diffraction. The authors speculate that their computationally
designed enzymes resemble primordial enzymes more than
highly refined modern-day enzymes. In any case, Jiang et al.
(2008) convincingly demonstrated that novel enzyme
activities can be designed from scratch through the use of
their systematic approach.
1.6.7 We Need to Define the Efficient Routes
for Obtaining High Potency Enzyme
Inhibitors as Drugs and Pesticides
Enzyme inhibitors are by far the most effective drugs,
because an inhibitor’s effect on metabolism is magnified by
the target enzyme’s catalytic efficiency. It’s also the case
that an enzyme’s specificity for its substrate(s) is often
manifested in its interactions with inhibitors.
Hopkins and Groom (2002) concluded that only about
3,000 of the 30,000 genes in the human genome can be
classified as ‘‘disease-modifying genes.’’ The ever-expanding enterprise of developing the next cadre of billion-dollar
drugs depends heavily on the discovery of new enzymes and
inhibitors that may serve as drug targets and as lead molecules that guide drug design. Most drug discovery efforts
begin with the recognition that a compound shows promise
as an inhibitor of an enzyme of pharmacologic interest. Such
molecules, called lead compounds (or simply leads), must
run the gauntlet of criteria for evaluating the promise of
a new drug. Capitalizing on mode-of-action information,
pharmacologists and medicinal chemists are perfecting
strategies for developing novel drugs (Copeland, 2005).
Combinatorial libraries of organic compounds are also
employed to identify leads based on the ability of randomly
shaped molecules to fill cavities within an enzyme’s active
site. Genomics and proteomics are likewise being explored
as new avenues for identifying lead molecules.
Chapter j 1 An Introduction to Enzyme Science
45
FIGURE 1.10 Candidate motifs for
catalysis
of
retro-aldol
reaction
mechanisms. Shown are active-site motifs
with quantum mechanically optimized
structures. Motif I, possessing two lysines
positioned nearby each other to lower the
pKa of the nucleophilic lysine, and a LysAsp dyad acting as the base to deprotonate
the hydroxyl group. Motif II, with catalytic
lysine buried in a hydrophobic environment
to lower its pKa, thereby increasing its
nucleophilic character, and a tyrosine that
can function as a general acid or base. HB,
hydrogen bond. (Top right) Motif III,
wherein the catalytic lysine (analogous to
Motif II) is in a hydrophobic pocket to
lower its pKa, and a His-Asp dyad serves as
a general base similar to the catalytic unit
commonly observed in the serine proteases.
Motif IV, with the catalytic lysine is again
positioned in a hydrophobic environment.
Additionally, an explicitly modeled bound
water molecule is placed, such that it forms
a hydrogen bond with the carbinolamine
hydroxyl during its formation, aids in the
water elimination step, and deprotonates the
b-alcohol at the C–C bond-breaking step. A hydrogen-bond donor/acceptor, such as Ser, Thr, or Tyr, is placed to position the water molecule in
a tetrahedral geometry with the b-alcohol and the carbinolamine hydroxyl. The proton abstracting ability of the water molecule is enhanced by a second
hydrogen bond with a base residue. We incorporated, where possible, additional hydrogen-bonding interactions to stabilize the carbinolamine hydroxyl
group and an aromatic side chain to optimally pack along the planar aromatic moiety of the substrate. Figure and legend adapted from Jiang et al.
(2008) are reproduced here with permission of the authors and the publisher.
The most reliable tools, by far, are the mechanistic
insights obtained through kinetic analysis of enzyme action,
and such efforts will doubtlessly require advances in
enzyme science as well as structural biology, molecular
mechanics, and physical biochemistry. A particularly
fruitful approach is to infer the most likely transition-state
geometry through the determination of kinetic isotope
effects. These concepts and experimental strategies are
described in Chapters 8 and 9.
1.6.8 We Need to Learn More About
In Singulo Enzyme Catalysis
Direct visualization of catalytic reaction cycles of an individual enzyme molecule (hence the term in singulo) is at
long last feasible. Enzyme kinetic experiments have traditionally been carried out with large numbers of enzyme
molecules, and even 1-nL volume of 1 nM enzyme contains
nearly a million molecules. Advances in protein science,
optics, fluorescence and solid-state electronics, however,
make possible the direct observations of single enzyme
molecules.
The ergodic hypothesis asserts that the time-average
of a physical quantity along a time trajectory of an
individual member within a homogeneous ensemble is
equivalent to the ensemble-averaged value of that
quantity at a given time (Gillespie, 1992; Norris, 1997;
van Kampen, 1992). A powerful justification for conducting single-molecule observations is the need to test
whether individual members are indeed representative of
the overall population of molecules (Xie and Trautman,
1998). Reaction trajectories can now be reliably determined for individual enzyme molecules that are physically isolated from each other by attachment to solid
surfaces or supramolecular structures, during confinement
within a gel or polymer matrix, or as they operate catalytically and move freely within an extremely small
volume element. As will become clear later in this
reference book, other breakthroughs in materials science
and chemical physics have also spurred the development
of single-molecule kinetics.
Enzyme chemists and statistical physicists are similarly
intrigued by the stochastics of enzyme catalysis and cooperativity (e.g., activity fluctuations, pausing, waiting-time
distributions, static disorder, fluctuating reactant concentrations, etc.). Such information affords the opportunity to
compare individual and ensemble-averaged properties
unambiguously, thereby bridging the microscopic and
macroscopic worlds of chemistry.
These concepts and the ever-expanding armamentarium
of experimental tools for testing them are explored more
fully in Chapter 12.
46
Enzyme Kinetics
FIGURE 1.11 Structures of computationally designed enzymes. A–C: Examples of design models for active site designs highlighting groups
important for catalysis. The nucleophilic imine-forming lysine is in orange, the transition-state model is in yellow, the hydrogen-bonding groups
are in light green, and the catalytic water is shown explicitly. The designed hydrophobic binding site for the aromatic portion of the TS model
is indicated by the gray mesh. A: RA60 (catalytic motif IV, jelly-roll scaffold), wherein a designed hydrophobic pocket encloses the aromatic
portion of the substrate and packs the aliphatic portion of the imine-forming Lys48. A designed hydrogen-bonding network positions the bridging
water molecule and the composite TS. B: RA46 (catalytic motif IV, TIM-barrel scaffold), wherein Tyr-83 and Ser-210 position the bridging water
molecule, thereby potentially facilitating required proton shuffling in active site IV. C: RA45 (catalytic motif IV, TIM-barrel scaffold). The bridging
water is hydrogen-bonded by Ser-211 and Glu-233; replacing the Glu-233 with Thr decreases catalytic activity by a factor of three. D and E: Overlay of design model (purple) on X-ray crystal structure (green). Designed amino acid side-chains are shown in stick representation, and the TS
model in the design is shown in gray. D: The 2.2 Å crystal structure of the Ser-210-Ala variant of RA22 (catalytic motif III, TIM-barrel scaffold).
The root mean square deviation (RMSD) for Ca atoms for the design model and its crystal structure is 0.62 Å, and the heavy-atom RMSD in the
active-site is 1.10 Å. E: 1.8 Å crystal structure of Met-48-Lys variant of RA61 (catalytic motif IV, jelly-roll scaffold). Design-crystal structure Caatom RMSD is 0.46 Å, and heavy-atom RMSD is 0.8 Å. The small differences in the high-resolution details of packing around the active site are
believed to arise from slight movements in some of the loops above the binding pocket and two rotamer changes in RA61 that may reflect the
absence of a bound TS analogue in the crystal structure. Figure and legend adapted from Jaing et al. (2008) are reproduced here with permission
of the authors and the publisher.
1.6.9 We Need to Develop Comprehensive
Catalogs of Enzyme Mechanisms and to Use
Such Information in Fashioning New
Metabolic Pathways
A promising development that should foster rational
comparison of enzyme reaction mechanisms and perhaps
even the design of new metabolic pathways is the
MACiE database (Holliday et al., 2005, 2006). This
internet-accessible bioinformatics database standing for
Mechanism, Annotation and Classification in Enzymes
(go to: http://www.ebi.ac.uk/thornton-srv/databases/MACiE/
glossary.html) categorizes the reaction mechanisms of
well-characterized enzymes in the Protein DataBase
(PDB). MACiE is a collaborative project between John
Mitchell’s Group at the Unilever Center for Molecular
Information at Cambridge University and Janet Thornton’s research group at the European Bioinformatics
Institute, located south of Cambridge. All curated
mechanisms are taken from the primary literature by
a suitably trained chemist and biochemist. Each enzyme
is assigned an identifying number based on the Enzyme
Chapter j 1 An Introduction to Enzyme Science
Commission system (go to: http://www.chem.qmul.ac.uk/
iubmb/enzyme/). The MACiE database specifies: (a) reaction identifier; (b) overall reaction type; (c) atoms involved;
(d) bonds involved; (e) bonds broken; (f) bonds made; (g)
substrates, cofactors products, along with suitable Kegg
Ligand Database identifiers (go to: http://www.genome.jp/
ligand/); (h) groups transferred; (i) groups eliminated;
(j) species reduced; and (k) species oxidized.
While admittedly more daunting, reaction stages are
annotated with respect to: (i) involved substrate, cofactor
and/or product; (ii) reaction centers; (iii) rate-determining
step? (iv) reversible step? (v) stage reaction type; (vi)
group(s) transferred; (vii) involved nucleophile; (viii) type
of tautomerization; (ix) reaction type; (x) reaction attributes; (xi) bond cleaved; (xii) bond formed; (xiii) bondorder change; and (xiv) involved residues, whether
a nucleophile, charge stabilizer, spectator, etc. As described
by Holliday et al. (2005; 2006), the process of annotating
the data contained within MACiE involves advanced
methods to minimize erroneous data entry. Wherever
possible, issues of semantics are resolved by reference to the
IUPAC Gold Book (go to: http://goldbook.iupac.org/) as
well as the MACiE dictionary (go to: http://www.mitchell.
ch.cam.ac.uk/macie/glossary.html). An added advantage
of MACiE is that it should become feasible to identify
known enzymes as best-case candidates for the generation
of novel catalysts via site-directed mutagenesis. Because
the overall reaction is treated as the composite of mechanistic steps, MACiE should eventually resolve shortcomings in the EC nomenclature of energase-class enzymes
(Purich, 2001).
As noted earlier, fully one-fifth of the gross national
product of an industrialized country depends on catalysis.
Unfortunately, most synthetic catalysts exploit special
properties of aluminum, chromium, manganese, nickel,
platinum, palladium, ruthenium, etc., of which most are
inherently toxic as elemental metals or simple metal
oxides. Techniques that increase their effective surface
area, such as atomic deposition on carbon or zeolites,
also increase their hydrolysis and undesired entry into
the biosphere. Given the significance of catalysis in our
everyday lives, it may be reasonably expected that
natural or ‘‘remanufactured’’ enzymes will play a major
role in efforts to develop a ‘‘Green Chemistry’’ that is
both efficient and ecologically sound. Because the
cardinal features of enzymes are specificity and high
turnover, and because enzymes are completely biocompatible, enzyme science has much to offer in the development of catalysts affording high yields and low
toxicity. For example, enzyme-catalyzed biofuel cells
may soon offer an alternative to transition metal catalysts
for power generation. They could, in principle, facilitate
oxidize alcohols at relatively low over-potential without
the production of detrimental carbon monoxide, and are
capable of operation at lower temperatures. Palmore
47
et al. (1998) described a methanol/O2 biofuel cell that
uses an NADþ-dependent dehydrogenase as catalysts and
exploits an electro-enzymatic method to regenerate
NADH at modest over-potentials. We may also surmise
that effective photo-electro-enzymatic methods will
likewise harness solar energy to create electrode overpotentials.
Pointing to the overwhelming impact of human activity
on Earth’s biosphere, futurists tell us that thermal pollution is unavoidable. Some suggest that the effects of
global warming have been grossly underestimated, simply
because higher temperatures are suppressed by the buffering effects of deep ocean currents; once these heat sinks
are loaded, unchecked ‘‘temperature creep’’ may manifestly become what may be regarded as human-generated
heat. The only apparent counter-measure is inventive
conservation, where new efficiencies must be realized
through improved machine designs and/or where chemists
devise better ways to transduce solar energy into chemically stored energy. If chlorophyll is the answer,10 then
one or more enzymes will likewise play a part. If calciummediated depletion of CO2 is the answer, then the enzymology of biomineralization will enjoy mounting interest.
And if bacterial fermentation is the answer, new pathways
with enhanced enzymatic activities can be developed. U.S.
Patent Number 5,000,000, for example, describes a genetically engineered Escherichia coli that was transformed
with alcohol dehydrogenase and pyruvate decarboxylase
genes from Zymomonas mobilis (Ingram, Conway and
Alterthum, 1991). These genes are expressed at sufficient
levels to confer upon the resulting Escherichia coli
transformant an ability to produce ethanol fermentatively
at 80–90% efficiency. This patent shows that bacterial
enzymology is already playing a role in converting silage,
corn syrup, and even biodegradable landfill refuse into
biofuels.
Another fertile approach, pursued by Synthetic Genomics, Inc., is the design of entirely novel metabolic pathways using microorganisms that possess synthetic, or
stripped-down, genomes that are optimized to allow for
single-purpose production of valuable substances, biofuels,
etc. The goal is to modify the operating system of a cell to
direct the synthesis of metabolic products with commercial
value and improve those cellular properties essential for
large-scale commercial bioprocesses.
10
The following simple calculation indicates that an artificial system with
an efficiency comparable to photosynthesis would be a considerable source
of renewable energy. In the U.S., ~2500 hours per year of sunlight reach
an intensity of ~800 watts per square meter, meaning that one hectare
(104 m2) receives ~2 1010 watt-hours of energy. If 50% of this solar
energy could be harvested as H2, the energy output would be ~1010
watt-hours of energy.
48
1.6.10 We Need to Understand How to
Analyze the Kinetic Behavior of Discrete
Enzyme-Catalyzed Reactions as Well as
Metabolic Pathways in their Environment
Our knowledge about how individual enzymes actually
operate within cells is surprisingly meager. Systematic
investigation of the intracellular kinetics of enzymes
promises to enrich our understanding of discrete enzymatic
processes as well as the flow of metabolic information that
is encoded in the ligand binding kinetics and enzymic
processes associated with signal transduction cascades.
Enhanced understanding intracellular enzyme kinetics
promises to improve the ways in which drugs are designed
and used, including efforts to minimize harmful sideeffects.
While we might anticipate that the availability of highresolution microscopes and high-sensitivity color cameras
would facilitate studies of enzymatic kinetics within living
cells, little progress has been made on measuring enzyme
kinetics in situ. A major challenge is that spectral signals
from substrates and products for an individual enzyme
reactions are most often obliterated by spectral signals from
the many chromophores and fluorophores of numerous
other metabolites. Consider, for example, the conversion of
NADþ, which itself is virtually transparent at 340 nm, to
NADH, which strongly absorbs 340-nm light. The problem
is that NADþ and NADH are involved in so many oxidoreductase reactions that cannot be uniquely associate an
absorbance change with a particular enzyme-catalyzed
reaction. The only exception is the use of synthetic chromogenic and fluorogenic alternative substrates in place of
their natural counterparts. Another challenge is that the
concentration of a particular enzyme may vary within
different subcellular regions. Living cells are also of irregular thickness, making it impossible to apply Beer’s Law
(i.e., Abs ¼ 3cl). Likewise, fluorescence measurements are
confounded by light scattering, quenching, as well as innerfilter effects.
Stable and radioactive isotopic tracers (see Chapters 4, 9,
and 11) are most often the best ways to analyze metabolic
flux, Ji, which is the net reaction rate (units ¼ DMolarity/Dt),
through the ith step in a pathway. Except for the rare instances
where a gaseous metabolite (e.g., CO2, CO, H2, CH4, NO, or
N2) is assimilated or released, isotopic assays are rarely
continuous, and substrate consumption or product formation
must be determined by mass spectrometry or liquid scintillation counting after a sample of cells is fixed, extracted, and
separated. In a few cases, NMR can be used if the labeled
species is present in sufficient quantities. The specialized field
known as Metabolic Control Analysis focuses on the
measurement of metabolic fluxes to learn how integrated
metabolic networks operate within its cellular context. In
MCA, the researcher seeks to understand large-scale
Enzyme Kinetics
dynamics of metabolic and physiological systems through
modeling and simulation that is cast in terms of the sensitivity
or responsiveness of metabolic flux to input signals. Metabolic Control Analysis is introduced in Section 11.13.
Whether such efforts successfully reproduce an enzyme’s intracellular interactions is largely a matter for
conjecture. Recognizing that the intracellular milieu may
alter the kinetic behavior of enzymes, some investigators
have conducted in vitro kinetics using suspensions of permeabilized cells to eliminate barriers to intracellular action
of an enzyme on substrate(s) supplied externally. The basic
approach is to disrupt the peripheral membrane by multiple
freeze-thaw cycles or by treatment with agents like digonin,
filipin, Triton X-100, or Lubrol WX. The goal is to allow
free access of low-molecular-weight substrates and metabolic effectors to enzymes within treated cells without dislodging the enzyme of interest from its normal site and
certainly without loss of proteins from the permeabilized
cells. A good system is the yeast Saccharomyces cerevisiae,
the cell wall of which, even after peripheral membrane
permeabilization, acts as a semipermeable barrier that
retains intracellular proteins while permitting small molecules to enter or leave (Chow and Palecek, 2004; Serrano,
Ganceda and Ganceda, 1973).
Students of muscle contraction long ago recognized the
power of cell permeabilization in managing the kinetics of the
actomyosin (AM) mechanochemical cycle and in investigating the action of myosin light chain kinase in the contractile
process. Both processes are ATP-dependent, and radioactive
ATP and/or photo-caged ATP (see Section 10.6.1) can be
supplied exogenously to suitably permeabilized muscle fibers.
He et al. (1997), for example, measured the rate of inorganic
phosphate (Pi) release, and hence overall ‘‘ATPase’’ activity of
rabbit psoas muscle in single, permeabilized muscle fibers that
were in rigor prior to laser flash photolysis of caged ATP in the
presence and absence of Ca2þ. The rate of Pi release from
AM$ADP$Pi complex was likewise monitored, based on the
rise in the fluorescence signal of the Pi-sensitive probe formed
by covalent labeling of bacterial phosphate-binding protein
with the reporter group MDCC (see Section 4.5). Use of the
permeabilized muscle fiber approach also affords the opportunity to pre-load myosin’s active sites with the nonhydrolyzable analogues p(NH)ppA and p(CH2)ppA in order to
study hydrolysis-sensitive steps in the AM reaction cycle.
Because most of our knowledge of regulatory molecule
interactions is the result of painstaking in vitro reconstitution experiments using fractionated cell components,
Mura and Stadtman (1981) opted to use permeabilized
bacterial cells to re-investigate the bicyclic protein
nucleotidylation cascade that was first discovered in
Stadtman’s laboratory (see Section 11.11). This prototypical system for enzyme-catalyzed reversible covalent
interconversion regulates the interconversion of dodecameric glutamine synthetase between its adenylylated
Chapter j 1 An Introduction to Enzyme Science
(catalytically active) and unadenylylated (catalytically
inactive) forms (Adler, Purich and Stadtman, 1975;
Stadtman and Ginsburg, 1974). At high concentration,
ammonia suffices for glutamine in numerous amido-synthase reactions leading to such nitrogenous metabolites as
histidine, N-acetyl glucosamine, and CTP. Earlier studies
with isolated protein and enzyme components indicated
that the state of glutamine synthetase adenylylation
depended on indicators of ammonia availability: a-ketoglutarate was found to be a signal for low ammonia
availability, whereas glutamine was an indicator that
ammonia was plentiful. Mura and Stadtman (1981) found
that permeabilization of Escherichia coli cells resulted in
complete retention of all protein components, presumably
the result of the bacterium’s Gram negative peptidogycan
cell wall. They found that the state of glutamine synthetase within permeabilized cells increased to a high state of
adenylylation in the presence of ATP and glutamine, with
~11 of the synthetase’s 12 subunits containing an Otyrosyl-AMP moiety. However, in the presence of a ketoglutarate, Pi, and ATP, the average number of O-tyrosylAMP residues decreased to ~2. Time-dependent changes
in the state of adenylylation that occur during incubations
of permeabilized cells in buffers containing these effectors
can be arrested either by sonication in the cold or by the
addition of cetyl-trimethyl-ammonium bromide (to inactivate adenylyltransferase). Mura and Stadtman (1981)
thus established that Lubrol-permeabilized cells are
a reliable way to investigate the regulation of glutamine
synthetase adenylylation in situ.
Given the need for additional approaches for investigating intracellular enzyme kinetics, it should be possible
to first permeabilize tightly adhered cells and then use an
over-layer of mineral oil to physically isolate each cell from
the others. A chromogenic or fluorogenic substrate could
then be micro-injected into the small volume of buffer
surrounding a cell of interest, and the progress of the reaction could then be sensed by absorption or fluorescence
spectroscopy.
1.6.11 We Need to Develop Techniques
that Will Facilitate Investigation of
Chromosomal Remodeling, Epigenetics,
and the Genetic Basis of Disease and Cell
Survival
Few fields within the broad scope of the molecular life
sciences are developing as rapidly as the fields of chromosomal remodeling and epigenetics. Recent thrusts in
molecular genetics, for example, led to the discovery of
many novel chromatin-associated enzymes, including:
numerous DNA methylases, which are responsible for
epigenetic marking; NAD+-dependent histone deacetylase,
49
which requires the unprecedented stoichiometric cleavage
of the redox coenzyme to facilitate amide hydrolysis;
telomerases, which add stabilizing DNA repeats (e.g.,
TTAGGG in vertebrates) to chromosome ends; NAD+dependent poly-ADPR polymerases (or PARPs), which
likewise modify chromosome stability; as well as a battery
of scores to hundreds of ATP hydrolysis-dependent,
chromatin-remodeling mechanoenzymes. Although genomics provides an upper bound on the likely number of
unique enzymes, there is no reliable metric for quantifying
the complexity of interactions among these catalysts and
their many protein, nucleic acid, and low-molecularweight metabolic effectors. In epigenetics, for example,
we are only beginning to glimpse how individual tissues
change during development, aging, and senescence to
modify the set-points for energy metabolism or how
epigenetic marks are maintained within an organism or
how these epigenetic marks undergo multi-generational
transmission from parent to child, and to succeeding
generations. And when long-range gene regulation is
considered (e.g., the multiple gene-coordinating action of
the locus control region (LCR) for stage-specific expression of hemoglobin genes within conceptus, fetus,
neonate, and adult), the likely pivotal importance of ATPdependent mechanoenzymes in prying open appropriate
highly compacted chromatin regions for active transcription, while simultaneously limiting RNA polymerase
access to those genes that must remain quiescent, cannot
be overstated.
We may likewise anticipate that a strong collaboration
among enzyme kineticists and molecular geneticists will
also quicken the pace of discovery of novel chromosome
regulation at this dawning hour. Learning how and when
various chromatin-remodeling mechanoenzymes find and
interact with specific locations within the nucleus also
promises to provide the opportunity to alter cell function
and/or proliferation. Indeed, timely development of novel
kinetic assays allowing one to probe the in situ action of
gene-regulating enzymes is of paramount importance. So
also will be the rationale design of novel inhibitors that
are based on systematic investigation of the kinetic and
catalytic mechanisms of these enzymes. For example,
systematic kinetic isotope effect studies on the NADþdependent histone deacetylases and telomerases should
provide valuable clues about the transition-state structure
and its acid/base properties. As described in Sections
8.6.1, 8.12.4, and 9.6, such information is essential for
the design of high-affinity transition-state inhibitors.
Ultimately, collaborations among enzyme kineticists and
molecular geneticists will also enlarge the tally of new
druggable target enzymes, thereby expanding the
opportunity to develop a wider spectrum of drugs and
therapeutic regimens that should improve the health,
performance, and sustained vitality of plants and
animals.
Enzyme Kinetics
50
1.6.12 We Need to Develop Effective
Enzyme Preparations for Use in Direct
Enzyme Therapy
The speed and specificity of enzyme catalysis commends
direct enzyme therapy (i.e., the use of small quantities of
certain enzymes as drugs to treat patients by modifying
metabolism and/or ridding cells of disease-producing
metabolites or toxins). This strategy includes and goes
beyond enzyme replacement therapy, wherein a deficient,
inactive, or absent enzyme is replaced by gene therapy and,
less often, by infusion. The potential of direct enzyme
therapy was first entertained over 50 years ago by Linus
Pauling, who was the first to trace a molecular basis of
a disease (sickle cell anemia) to a likely amino acid
substitution (later shown to be the Glu-to-Val mutation
position-6 within the b-hemoglobin chain). For Pauling, the
objectives for direct enzyme therapy were deceptively
simple – identify a disease-causing enzyme defect or deficiency and replace that enzyme with one having full catalytic and/or regulatory capacity.
Table 1.5 presents those cases in which direct enzyme
therapy has been achieved or is nearing realization. Despite
many determined efforts, the successes are still far too few,
inviting the question: What limits the use of enzymes as
direct therapeutic agents? To address this issue, we may
first categorize therapeutic enzymes as those autologous
enzymes – those that are already normally produced by
healthy subjects within a given species versus heterologous
enzymes – those that originate in a different species. These
categories may be further subdivided on the basis of
whether an enzyme normally operates within or outside
the confines of a cell. Autologous extracellular enzyme
replacement offers greatest promise, because these enzymes
should exhibit limited immunogenicity, low toxicity, and
should already be well adapted to the inherently oxidizing
environment outside cells. For those enzymes destined for
use in intracellular therapy, the researcher must overcome
the additional obstacle of delivering the enzyme to the
correct intracellular compartment as well as in a physiologically controlled concentration range. The use of foreign
enzymes increases the likelihood that the host cells will
exhibit apoptotic instability and that the enzyme may
undergo rapid turnover. By far, the greatest obstacles for the
clinical efficacy of intracellular enzyme therapy will be
specific or selective delivery of the enzyme to the proper
cell/tissue target(s) and in the proper dosage. Except in
rare circumstances, expression vectors like adenovirus,
adeno-associated virus, and lentivirus are rarely delivered
with adequate specificity, and surface expression of viral
TABLE 1.5 Selected Examples of Direct Enzyme Therapy
Adenosine deaminase
Asparaginase and glutaminase
Collagenases
Dermal RNases
DNase
a-Galactosidase A
Glucocerebrosidase
a-Glucosidase
Lactase
Lecithinized superoxide dismutase
Lysozyme
Onconase (RNase)
Oxalate decarboxylase
Phenylalanine ammonia lyase
Proteases
Corrects adenosine deaminase-linked severe combined immune deficiency (ADA-SCID), by
preventing accumulation of toxic metabolites that impair cellular and humoral immunity.
Reduces the viability of asparagine- and glutamine-requiring tumor cells by hydrolyzing
asparagine and glutamine.
Debrides skin lesions, including scar tissue, ulceration, burns, and infected blisters.
Inhibits RNase-sensitive organisms, when applied in conjunction with membrane-lyzing
detergents. (Importantly, dermal RNase activity is not blocked by 5’-capping of mRNA.)
Treats chronic bronchitis by reducing bronchial mucous viscosity (a) by hydrolyzing DNA
and (b) by forming high-affinity complex with actin monomers, thereby greatly reducing
level of filamentous actin.
Treats a variety of clinical manifestations of Fabry’s disorder by reducing
globotriaosylceramide that accumulates in different cell types.
Treats Gaucher’s disease, which is by far the most common lysosomal storage disease.
Ameliorates late-onset Type 2 Glycogen Storage (or Pompe) Disease, a progressive multisystem disease evoked by a deficiency of lysosomal acid a-glucosidase.
Relieves gastrointestinal distress, flatulance, as well as skin lesions in 75% of all adults
worldwide who metabolize lactose poorly.
Ameliorates severe hypovolemia caused by increased blood vessel permeability following
burns by using its lecithin group to bind securely to dermal membranes, thereby allowing
destruction of surface superoxide.
Prevents microbial overgrowth by lyzing cell walls of various human pathogens.
Treats cancer A by triggering apoptosis as a consequence of messenger RNA and micro RNA
degradation.
Reduces renal calcium oxalate monohydrate stone formation by decomposing dietary
oxalate.
Treats phenylketonuria by reducing serum levels of phenylalanine, which is converted to
toxic phenylpyruvate.
Treats bacterial infection by hydrolyzing pathogen cell walls and microbial biofilms. Some
preparations also reduce HIV infection.
Chapter j 1 An Introduction to Enzyme Science
antigens raises the specter of cellular immunity and
apoptosis. A highly efficient means for incorporating
enzymes and other proteins into cells is afforded by adding the membrane-penetrating (or penetratin) sequences
RQIKIWFQNRRMKWKK and RRRQRRKKR, found
respectively within Drosophila antennapedia and HIV-TAT
proteins, to the primary sequence of potential therapeutic
enzymes. These sequences allow rapid and direct incorporation of proteins into the cytoplasm of all cells tested to
date. Even so, delivery to the proper cell target remains
problematical. Whether delivered by means of viral vectors
or as penetratin-containing fusion enzyme, the elusive goal
of maintaining enzyme dosage within a narrow well
controlled range represents the Holy Grail for direct enzyme
therapy.
As potential therapeutic enzymes are identified and
developed, we can be reasonably certain that site-directed
mutagenesis and chemical modification (e.g., conjugation to
polyethylene glycol for reduced immunogenicity or to
lecithin or by recombinant methods to introduce CAAXtype acylation sequences for enhanced membrane docking)
will be essential tools for adapting these enzymes for clinical use. As will be discussed in Section 7.15.4, every sitedirected enzyme mutant must also be treated as though it is
an entirely new enzyme, each potentially with its unique
physical and chemical properties. The same may be said for
chemical modified enzymes. Such statements point to the
need for substantial kinetic characterization of these
modified enzymes to verify their likely effectiveness. Also
required are appropriate kinetic tests of the efficiency of
enzyme dissolution and dispersion when formulated
enzymes are introduced into blood or model cell types as
well as kinetic measurements of enzyme turnover.
51
FURTHER READING
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Altman, S. (1989). Ribonuclease P: an Enzyme with a Catalytic RNA
Subunit. Adv. Enzymol., 62, 1.
Cech, T. R. (Ed.). (1993). The RNA World. Cold Spring Harbor, New York:
Cold Spring Harbor Press. pp. 239.
Copeland, R. A. (2005). Evaluation of Enzyme Inhibitors in Drug
Discovery: A Guide for Medicinal Chemists and Pharmacologists.
Hoboken: Wiley-Interscience. pp. 271.
Frey, P. A., & Hegeman, A. D. (2006). Enzymatic Reaction Mechanisms.
New York: Oxford University Press. pp. 768.
Guerrier-Takada, C., Gardiner, K., Maresh, T., Pace, N., & Altman, S.
(1983). The RNA Moiety of Ribonuclease P is the Catalytic Subunit
of the Enzyme. Cell, 35, 849.
Hammes, G. G. (2002). Multiple Conformational Changes in Enzyme
Catalysis. Biochemistry, 41, 8221.
Haldane, J. B. S. (1930). Enzymes. London: Longmans-Green.
Jencks, W. P. (1969). Catalysis in Chemistry and Enzymology. San Francisco: McGraw-Hill.
Metzler, D. E. (2004). Biochemistry: The Chemical Reactions of Biological Systems. New York: Academic Press.
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Purich, D. L., & Allison, R. D. (2002). The Enzyme Reference. New York:
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