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Enzymes
It's true hard work never killed anybody, but I figure, why take the chance? Ronald
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Reagan
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ENZYMES
Thermodynamic principles can be used to indicate whether or
not a reaction can take place spontaneously
They do not, however, provide information about the rate at
which a reaction will proceed
Most biochemical reactions proceed so slowly at physiological
temperatures that catalysis is essential for the reactions to
proceed at a satisfactory rate in the cell
At temperatures above absolute zero, all molecules possess
vibrational energy which increases as the molecules are heated
As the temperature rises, vibrating molecules are more likely to
collide
A chemical reaction occurs when colliding molecules possess a
minimum amount of energy called the activation energy
Not all collisions result in chemical reactions because only a
fraction of the molecules have sufficient energy or the correct
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orientation to react
• Another way of increasing the likelihood of collisions, thereby
the formation of product, is to increase the concentration of
reactants
• In living systems, however, elevated temperatures may harm
delicate biological structures and reactant concentrations are
usually quite low
• The preferred catalysts in living systems, therefore, are
enzymes, most of which are proteins (except for ribozymes
which are RNA)
• Enzymes can increase the rate of a reaction by several orders
of magnitude
• Enzymes do their job by decreasing activation energy,
thereby increasing the percentage of substrate molecules
that have sufficient energy to react
• Indeed, in the absence of enzymes, life as we know it would
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not be possible
The Effect of Catalysis on
the Activation Energy of a Reaction
• Enzymes do not affect ΔG (the position of the equilibrium) but
speed up its attainment. The concentrations of substrate and
product at equilibrium are not changed
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The Basic Features of Enzymes
1. Catalytic power
• Enzymes accelerate reaction rates as much as 1016 over
uncatalyzed levels, which is far greater than any synthetic
catalysts can achieve
• And enzymes accomplish these astounding feats in dilute
aqueous solutions under mild conditions of temperature and pH
2. Specificity
• The action of enzymes is usually very specific. This applies not
only to the type of reaction being catalyzed (reaction
specificity), but also to the nature of the substrates that are
involved (substrate specificity)
• Enzymes with low reaction specificity and low substrate
specificity are very rare
• Intimate interaction between an enzyme and its substrates
occurs through molecular recognition based on structural
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complementarity
• Such mutual recognition is the basis for specificity. The
specific site on the enzyme where substrate binds and
catalysis occurs is called the active site
3. Regulation
• Although the enormous catalytic potential is essential, it
does pose a problem: if it was not regulated, all reactions in a
cell would rapidly reach equilibrium and, once again, life as
we know it would not be possible
• Regulation of enzyme activity is achieved in a variety of ways,
ranging from controls over the amount of enzyme protein
produced by the cell to more rapid, reversible interactions of
the enzyme with metabolic inhibitors and activators
 There should be a system for the classification and naming of
the several enzymes present in the cell
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Enzyme Nomenclature
The commonly used names for most enzymes describe the
type of reaction catalyzed, followed by the suffix –ase
For example, dehydrogenases remove hydrogen atoms,
proteases hydrolyze proteins and isomerases catalyze
rearrangements
Modifiers may precede the name to indicate the substrate
(lysyl oxidase), the source of the enzyme (pancreatic
ribonuclease), its regulation (hormone-sensitive lipase) or a
feature of its mechanism of action (cysteine protease)
Where needed, alphanumeric designators are added to
identify multiple forms of an enzyme (eg, RNA polymerase III;
protein kinase Cβ).
To address ambiguities, the International Union of
Biochemists (IUB) developed an unambiguous system of
enzyme nomenclature
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• In this system, each enzyme has a unique name and code
number that identify the type of reaction catalyzed and the
substrates involved
• Although common names for many enzymes remain in
use, all enzymes now are classified and formally named
according to the reaction they catalyze
• Six classes of reactions are recognized. Within each class
are subclasses, and under each subclass are subsubclasses
within which individual enzymes are listed
• Classes, subclasses, subsubclasses and individual entries
are each numbered, so that a series of four numbers serves
to specify a particular enzyme –Enzyme Commission (EC)
Number
• A systematic name, descriptive of the reaction, is also
assigned to each entry
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Classes of Enzymes
1. Oxidoreductases
• Oxidative reactions remove electrons, usually one or two
electrons per molecule of substrate, while reductive reactions
accomplish the converse
• Oxidoreductases transfer electrons from one compound to
another, thus changing the oxidation state of both substrates
• In many oxidation reduction reactions, hydrogen is transferred
along with electrons
• In other reactions, a molecule or atom of oxygen could be
transferred to a substance; electrons could also be transferred
to oxygen
2. Transferases
• Transferases catalyze reactions in which a functional group is
transferred from one compound to another
• Commonly transferred functional groups include phosphate,
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amino, methyl
3. Hydrolases
• Cleave carbon-oxygen, carbon-nitrogen, carbon-sulfur,…
bonds by adding water across the bond
• Digestive enzymes are hydrolases
4. Lyases
• Cleave carbon-oxygen, carbon-nitrogen, carbon-sulfur,…
bonds but do so without addition of water and without
oxidizing or reducing the substrates
• Double bonds either arise or disappear through the action of
lyases
5. Isomerases
• Catalyze intramolecular rearrangements of functional
groups that reversibly interconvert optical or geometric
isomers
• When an isomerase catalyzes an intramolecular
rearrangement involving movement of a functional group, it
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is called a mutase; what is a racemase?
6. Ligases
• Catalyze biosynthetic reactions that form a covalent bond
between two substrates.
• Ligases differ from lyases in that they utilize the energy
obtained from cleavage of a high-energy bond to drive the
reaction. The molecule with the high-energy bond is usually ATP
• Ligases are sometimes known as synthetases and lyases ,
synthases
• Because the systematic names were frequently cumbersome
and the numbers difficult to memorize, the EC also proposed
that a single recommended (trivial) name should be retained
(or invented) for each enzyme
• For example, the enzyme catalyzing the reaction:
ATP+AMP<----->2ADP
bears the systematic name ATP : AMP phosphotransferase,
the number EC 2.7.4.3 and the recommended name,
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adenylate kinase; the earlier name, myokinase, was forsaken
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Cofactors
Many enzymes carry out their catalytic function relying solely
on their protein structure
Many others require non-protein components, called
cofactors
Cofactors may be metal ions or organic molecules referred to
as coenzymes
Cofactors, because they are structurally less complex than
proteins, tend to be stable to heat. Typically, proteins are
denatured under such conditions
Usually coenzymes are actively involved in the catalytic
reaction of the enzyme, often serving as intermediate carriers
of functional groups in the conversion of substrates to
products
In many cases, a cofactor is firmly associated with its enzyme,
through covalent or non-covalent bonds. Such tightly bound
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cofactors are referred to as prosthetic groups of the enzyme
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Enzymes in which metal ions serve as prosthetic groups are
called metalloenzymes. Enzymes that require a non-bound
metal ion cofactor are termed metal-activated enzymes
The catalytically active complex of protein and prosthetic
group is called the holoenzyme. The protein without the
prosthetic group is called the apoenzyme; it is catalytically
inactive
Many coenzymes are vitamins or contain vitamins as part of
their structure
Vitamins are small organic molecules that are not
synthesized in the body and are therefore essential dietary
nutrients
The vitamins that are coenzyme precursors or coenzymes
include all the water-soluble B vitamins, vitamin C and the
fat-soluble vitamin K
Many coenzymes contain, in addition, the adenine, ribose
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and phosphoryl moieties of AMP or ADP
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Coenzymes are typically modified by certain reactions and are
then converted back to their original forms by other enzymes;
small amounts of these substances can be used repeatedly
Vitamin B1: Thiamine
• It is the precursor of thiamine pyrophosphate (TPP), a
coenzyme involved in reactions where bonds to carbonyl
carbons (aldehydes or ketones) are synthesized or cleaved
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Niacin (nicotinic acid): Vitamin B3
• Nicotinamide is an essential part of two important
coenzymes: nicotinamide adenine dinucleotide (NAD+) and
nicotinamide adenine dinucleotide phosphate (NADP+)
• The reduced forms of these coenzymes are NADH and
NADPH
• The nicotinamide coenzymes (also known as pyridine
nucleotides) are electron carriers. They play vital roles in a
variety of enzyme–catalyzed oxidation–reduction reactions
• NAD+ is an electron acceptor in oxidative (catabolic)
pathways and NADPH is an electron donor in reductive
(biosynthetic) pathways
• These reactions involve direct transfer of hydride anion (H:)
either to NAD(P) + or from NAD(P)H
• The hydride anion contains two electrons, and thus NAD +
and NADP + act exclusively as two-electron carriers
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• The C-4 position of the pyridine ring, which can either accept or
donate hydride ion, is the reactive center of NAD + and NADP +
• Humans can synthesize some amount of niacin from
tryptophan. However, if dietary intake of tryptophan is low,
nicotinic acid is required for optimal health
• Nicotinic acid, which is beneficial to humans and animals, is
structurally related to nicotine, a highly toxic tobacco alkaloid
• In order to avoid confusion of nicotinic acid and nicotinamide
with nicotine itself, niacin was adopted as a common name for
nicotinic acid
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The Structures and Redox
States of the Nicotinamide
Coenzymes
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Riboflavin: Vitamin B2
• Is a precursor of both riboflavin 5-phosphate, also known as
flavin mononucleotide (FMN), and flavin adenine
dinucleotide (FAD)
• The name riboflavin is a synthesis of the names for the
molecule’s component parts, ribitol and flavin
• The flavins have a characteristic bright yellow color and take
their name from the Latin flavus for “yellow”
• The oxidized form of the isoalloxazine structure absorbs light
around 450 nm (in the visible region) and also at 350 to 380 nm
• The color is lost, however, when the ring is reduced or
“bleached”
• Similarly, the enzymes that bind flavins, known as
flavoenzymes, can be yellow, red, or green in their oxidized
states. These enzymes also lose their color on reduction of the
bound flavin group
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The Structures of
FAD and FMN
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• Flavin coenzymes can exist in any of three different redox
states: fully oxidized flavin is converted to a semiquinone by a
one-electron transfer; a second one-electron transfer converts
the semiquinone to the completely reduced dihydroflavin
• The three different redox states allow flavins to participate in
one-electron transfer and two-electron transfer reactions
The Oxidation States of
FAD and FMN
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Panthotenic Acid: Vitamin B5
• Makes up one part of a complex coenzyme called
coenzyme A (CoA)
• Pantothenic acid is also a constituent of acyl carrier
protein (ACP)
• Coenzyme A consists of 3,5-adenosine bisphosphate
joined to 4-phosphopantetheine in a phosphoric anhydride
linkage
• As was the case for the nicotinamide and flavin
coenzymes, CoA also contains an adenine nucleotide
moiety
• CoA and ACP are involved in the activation and transfer of
acyl groups
• The functions of CoA are mediated by the reactive
sulfhydryl group on CoA, which forms thioester linkages
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with acyl groups
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Pyridoxine: Vitamin B6
• The biologically active form of vitamin B6 is pyridoxal-5phosphate (PLP)
• PLP participates in the catalysis of a wide variety of reactions
involving amino acids, including transaminations,
decarboxylations, racemizations and eliminations
• PLP is found as a prosthetic group attached to enzymes
through a Schiff base formed between its aldehyde group and
the ε-amino group of a lysine residue
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The Tautomeric Forms of PLP
PLP Attached to an Enzyme
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Biotin : Vitamin B7
• Acts as a mobile carboxyl group carrier in a variety of
enzymatic carboxylation reactions
• In each of these, biotin is bound covalently to the enzyme as a
prosthetic group via the ε-amino group of a lysine residue on
the protein
• The biotin-lysine function is referred to as a biocytin residue
The result is that the biotin ring system is tethered to the
protein by a long, flexible chain
The Biocytin Complex
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Folic Acid: Vitamin B9
• Folic acid derivatives (folates) are acceptors and donors of onecarbon units for all oxidation levels of carbon except that of
CO2 (where biotin is the relevant carrier)
• The active coenzyme form of folic acid is tetrahydrofolate
(THF)
• THF is formed via two successive reductions of folate by
dihydrofolate reductase
Folic Acid
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THF
Cyanocobalamin: Vitamin B12
• Cyanocobalamin is converted in the body into two coenzymes:
the predominant coenzyme form is 5-deoxyadenosylcobalamin,
but smaller amounts of methylcobalamin also exist
• The corrin ring, with four pyrrole groups, is similar to the heme
prophyrin ring, except that two of the pyrrole rings are linked
directly; iron is substituted by cobalt
• There are two reactions in the body in which vitamin B12 is
known to participate: a molecular rearrangement and a methyl
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transfer
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Vitamin B12 and its Coenzyme Forms
Ascorbic acid: Vitamin C
• Has the simplest chemical structure of all the vitamins; and the
coenzyme form is the vitamin itself
• Ascorbic acid functions as an electron carrier. It is a strong
reducing agent
• It is used in the regeneration of the active form of enzymes and
antioxidants
(+) H.
(-) 2H.
(-) H.
(+) 2H.
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The Lipid-Soluble Vitamins
• Vitamin K is the only fat-soluble vitamin with coenzyme role
The Vitamin A group
• Vitamin A or retinol often occurs in the form of esters, called
retinyl esters. The aldehyde form is called retinal or
retinaldehyde
• Retinol can be absorbed in the diet from animal sources or
synthesized from β-carotene from plant sources
• The aldehyde group of retinal forms a Schiff base with a lysine
on opsin, to form light-sensitive rhodopsin
The Vitamin D group
• The two most prominent members of the vitamin D family are
ergocalciferol (vitamin D2) in plants and cholecalciferol
(vitamin D3) in animals
• Cholecalciferol is produced in the skin of animals by the action
of ultraviolet light (sunlight, for example) on its precursor
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molecule, 7-dehydrocholesterol
Rhodopsin
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• Because humans can produce vitamin D3, “vitamin D” is not
strictly speaking a vitamin at all
• Retinol and cholecalciferol are actually prohormones (precursors
of hormones) that regulate transcription of DNA, and thus gene
expression
Tocopherol: Vitamin E
• α-tocopherol is a potent antioxidant; and once it has been
oxidized, it can be regenerated by vitamin C
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Naphthoquinone: Vitamin K
• Clotting factors such as thrombin undergo a post-translational
modification that involves the carboxylation of glutmate
residues
• γ-carboxyglutamyl residues are effective in the coordination of
calcium, which is required for the coagulation process
• The enzyme responsible for this modification, glutamyl
carboxylase, requires vitamin K for its activity
The Structure of the K Vitamins
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• Not all coenzymes are derived from vitamins
 Tetrahydrobiopterin (BH4), the coenzyme for hydroxylation
reactions of aromatic amino acids is synthesized in the body
from GTP (guanosine triphosphate)
 Lipoic acid is a coenzyme used to couple acyl transfer with
electron transfer
• Lipoic acid exists as a mixture of two structures: a closed-ring
disulfide form and an open–chain reduced form. Oxidation–
reduction cycles interconvert these two species
• Lipoic acid is found attached with a lysine residues on
enzymes
• Metal ions, which have a positive charge, contribute to the
catalytic process by acting as electrophiles . They assist in
binding of the substrate or they stabilize developing anions in
the reaction. They can also accept and donate electrons in
oxidation-reduction reactions
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Examples of metal ions and the enzymes they function with
include:
 Zn2+ : alcohol dehydrogenase, carbonic anhydrase
 Mg2+ : ATP-dependent reactions such as hexokinase
 Fe3+ and Cu2+: components of the enzymes of the
mitochondrial electron transport chain to the ultimate
electron acceptor, oxygen
The Different forms of Lipoic Acid
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How Do Enzymes Work?
• There are three characteristics of enzymes that form the basis
of most of their properties:
1. The Active Site
• In an enzyme, folding brings together amino acids, most of
which are not adjacent in the primary sequence, so that some
amino acids form a three-dimensional structure that binds
with the substrate to form the enzyme-substrate complex
• This complex results in catalysis
• The remainder of the amino acids in the enzyme are involved
in maintenance of the three-dimensional structure of the
enzyme, attaching the enzyme molecule to intracellular
structures (e.g. membranes) or in binding molecules (e.g.
allosteric effectors) that regulate the activity of the enzyme
2. The Enzyme-Substrate Complex
• Enzymes bind substrates to produce an enzyme-substrate
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complex as follows:
Amino Acid Side-Chain Groups Involved in Binding
NAD+ at the Active Site of an Enzyme
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k1
E+S k2
⇌ES
• Weak bonds, generally non-covalent ones, are involved in
formation of the complex, so that the reaction is readily
reversed
• The rate of the forward reaction is given by the concentration of
substrate multiplied by the rate constant k1, and rate of the
reverse reaction is given by the concentration of the product
multiplied by the rate constant k2
• The dissociation constant for the ES complex is k2/k1
• This is analogous to the formation of other complexes: for
example receptor-hormone complex; receptorneurotransmitter complex; antibody-antigen complex
• Formation of the enzyme-substrate complex can occur only if
the substrate possesses groups that are in the correct threedimensional orientation to interact with the binding groups in
the active site
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• A ‘lock and key’ analogy (Emil Fischer) has been widely used to
explain specificity but it is inadequate because the formation of
the enzyme-substrate complex involves more than a steric
complementarity between enzyme and substrate
• Enzymes are highly flexible, conformationally dynamic
molecules, and many of their remarkable properties, including
substrate binding and catalysis, are due to this flexibility
• Realization of the conformational flexibility of proteins led
Daniel Koshland to hypothesize that the binding of a substrate
by an enzyme is an interactive process
• The shape of the enzyme’s active site is actually modified upon
binding S, in a process of dynamic recognition between enzyme
and substrate called ‘induced fit’
• Substrate binding alters the conformation of the protein, so
that the protein and the substrate “fit” each other more
precisely. The conformation of the substrate also changes as it
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adapts to the conformation of the enzyme
3. The Transition State
• In a chemical reaction, one stable arrangement of atoms (the
substrate) is converted to another (the product)
• As this change proceeds, the atoms pass through an unstable
arrangement, known as the transition state, which can be
thought of as the ‘halfway house’ between the substrates and
the products
• The relevance of the transition state to kinetics is that the
rate of the overall reaction depends on the number of
molecules in this state: the more molecules in the transition
state, the greater is the rate
• The role of an enzyme is to increase the number of molecules
in this state
• Enzymes increase the number of molecules in the transition
state through one or more of five mechanisms
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The Energy Level and the Structural
Feature of the Transition State
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Mechanisms of Enhancing the Rate of a Reaction
Since the transition state possesses the least stable electron
distribution, an agent capable of supplying or withdrawing
electrons to or from stable parts of a substrate in order to
destabilize it, accelerates the rate of a reaction
General acid base catalysis
Addition of a proton from an acid to a molecule can cause
an electron to be withdrawn from one part of the molecule
to the part which binds the proton
A base removes a proton from a molecule which will also
cause electron shifts
If these shifts favor the formation of the transition state,
the rate of the reaction increases
The active sites of enzymes possess side-chain groups of
amino acids that act as acids or bases
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• The contribution of these groups is greatly enhanced if they
act in a concerted manner so that, as an electron is withdrawn
from one part of the substrate, another is donated to a
different part
• This is possible only when the relevant groups in the active site
are held in precisely the correct orientation so as to interact in
this way with the substrate
• One of the more versatile side-chains in this respect is the
imidazole group of histidine. In one environment it can act as
an acid whereas, in another environment, the same group can
act as a base
• This can occur with two histidines in the same active site
• Acid base catalysis occurs on the vast majority of enzymes. In
fact, proton transfers are the most common biochemical
reactions
 Specific acid base catalysis occurs when only the protons and
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hydroxyls present in solution are used
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Amino Acids Involved in Acid Base Catalysis
2. Covalent catalysis (formation of an intermediate)
• Most enzymes bind their substrates in a non-covalent manner
but, for those that do bind covalently, the intermediate must
be less stable than either substrate or product
• Many of the enzymes that involve covalent catalysis are
hydrolytic enzymes; these include proteases, lipases,
phosphatases and also acetylcholinesterase
• A number of amino acid side chains, including all those that
participate in acid base catalysis and the functional groups of
some coenzymes can serve as nucleophiles in the formation of
covalent bonds with substrates. These covalent complexes
always undergo further reaction to regenerate the free enzyme
An Example of Covalent Catalysis
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An Example of Covalent Catalysis
3. Metal Ion Catalysis
• Metals, whether tightly bound to the enzyme or taken up from
solution along with the substrate, can participate in catalysis in
several ways
• Ionic interactions between an enzyme-bound metal and a
substrate can help orient the substrate for reaction or stabilize
charged reaction transition states
• Metals can also mediate oxidation-reduction reactions by
reversible changes in the metal ion’s oxidation state
• Nearly a third of all known enzymes require one or more metal
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ions for catalytic activity
4. Proximity and Orientation
• In a reaction involving two substrates, the two must come
together in order to react
• The chance of them doing so depends upon their
concentration in the solution: this is increased locally by
providing adjacent binding sites for each substrate within
the active site
• This can increase the effective concentrations of the
substrates about 1000-fold
• Even when a collision between two substrates occurs it is
unlikely that they will both be in the correct orientation for a
reaction to take place
• Another property of the active site is that it binds the
substrates in such a way that their orientation favors the
reaction, i.e., it facilitates electron shifts that favor
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formation of the transition state
5. Strain (Distortion)
• Enzymes that catalyze -lytic reactions that involve breaking a
covalent bond typically bind their substrates in a conformation
slightly unfavorable for the bond that will undergo cleavage
• The resulting strain stretches or distorts the targeted bond,
weakening it and making it more vulnerable to cleavage
The Catalytic Mechanism of the Aspartic Protease Family
(Example of Acid Base Catalysis)
• Enzymes of the aspartic protease family, which includes the
digestive enzyme pepsin, the lysosomal cathepsins and the
protease produced by HIV share a common catalytic
mechanism
• Catalysis involves two conserved aspartyl residues, which act
as acid-base catalysts
• In the first stage of the reaction, an aspartate functioning as a
general base (Asp X) extracts a proton from a water molecule,
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making it more nucleophilic
• The resulting nucleophile then attacks the electrophilic
carbonyl carbon of the peptide bond targeted for hydrolysis,
forming a tetrahedral transition state intermediate
• A second aspartate (Asp Y) then facilitates the decomposition
of this tetrahedral intermediate by donating a proton to the
amino group produced by rupture of the peptide bond
• The two different active site aspartates can act simultaneously
as a general base or as a general acid because their immediate
environment favors ionization of one, but not the other
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Carbonic Anhydrase and Metal Ion Catalysis
A zinc prosthetic group in carbonic anhydrase is coordinated in
three positions by histidine side-chains. The fourth
coordination position is occupied by water
Binding of water to zinc reduces pKa of water from 15.7 to 7
Zinc facilitates the release of a proton from a water molecule,
which generates a hydroxide ion that can act as a nucleophile
The carbon dioxide substrate binds to the enzyme's active site
and is positioned to react with the hydroxide ion
The hydroxide ion attacks the carbon dioxide, converting it
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into bicarbonate ion
• The catalytic site is regenerated with the release of the
bicarbonate ion and the binding of another molecule of water
The Catalytic Mechanism of Carbonic Anhydrase
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Factors that Change the Activity of an Enzyme
• The main factors that can change the catalytic activity of an
enzyme are concentrations of substrates, pH, temperature
and inhibitors
• The effects of these factors and the means by which they are
studied are usually described as enzyme kinetics
The Effect of Substrate Concentration
• The activity or rate of an enzyme (v) varies according to the
substrate concentration [S]. The relationship is hyperbolic:
 At very low substrate concentrations, the reaction
rate is approximately first order (i.e. activity increases
approximately linearly with increase in substrate
concentration)
 At very high substrate concentrations, the rate of reaction
approaches zero order (i.e. the increase in substrate
concentration has very little effect on the rate of reaction)
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This behavior is a saturation effect: when v shows no increase
even though [S] is increased, the system is saturated with
substrate. The physical interpretation is that every enzyme
molecule in the reaction mixture has its substrate-binding site
occupied by S
 At intermediate substrate concentrations, the order is
intermediate between zero and first order
Even though enzymes with a single substrate are considered
here, the same principles apply to enzymes with more than
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one substrate
• A hyperbolic curve is described by an equation of the form:
• In the case of an enzyme,
where a and b are constants
• Two questions:
1. What is the mechanism of catalysis that accounts for the
hyperbolic relationship?
2.What are the constants a and b?
• These questions were answered by the works of Michaelis and
Menten & Briggs and Haldane
• Michaelis and Menten’s theory was based on the assumption
that the enzyme, E, and its substrate, S, associate reversibly to
form an enzyme-substrate complex, ES:
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• This association/dissociation is assumed to be a rapid
equilibrium (hence the equilibrium model), and Ks is the
enzyme : substrate dissociation constant. At equilibrium,
• Product, P, is formed in a second step when ES breaks down to
yield EP; this step is slower
• E is then free to interact with another molecule of S
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• The interpretations of Michaelis and Menten were refined by
Briggs and Haldane, who postulated that the concentration of
ES quickly reaches a constant value
• ES is formed as rapidly from E+S as it disappears by its two
possible fates: dissociation to regenerate E+S, and reaction to
form E+P
• This assumption is termed the steady-state assumption and is
expressed as
• Two additional assumptions are made:
 In the reaction mixture, [S] is greater than [E].However, [S]
is not so large that all enzyme molecules are in the ES form,
but [S] must be sufficiently large that [S] does not rapidly
become so small that [S] < [E]
 Because enzymes accelerate the rate of the reverse reaction
as well as the forward reaction, it would be helpful to ignore
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any back reaction by which EP might form ES
The velocity of this back reaction would be given by v=k-2[E][P].
However, if only the initial velocity for the reaction (immediately
after E and S are mixed in the absence of P) is measured, the rate
of any back reaction is negligible because its rate will be
proportional to [P], and [P] is essentially 0
• The total amount of enzyme is fixed and is given by the formula
total enzyme, [ET]=[E]+[ES] where [E] = free enzyme and
[ES]=the amount of enzyme in the enzyme– substrate complex
• The rate of [ES] formation is
• The rate of disappearance of ES
• At the steady state, rate of formation=rate of disappearance
• Rearranging gives
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• The ratio of constants (k-1+k2)/k1 is itself a constant and is
defined as the Michaelis constant, Km
• Because it is given as a ratio of concentrations, the unit of Km is
molarity
• Rearranging the equation for the derivation of Km
• The rate of product formation is given as
v=k2[ES]
• Substituting for the value of [ES]
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• The product k2[ET] has a special meaning. When [S] is high
enough to saturate all of the enzyme, the velocity of the
reaction, v, is maximal
• At saturation, the amount of [ES] complex is equal to the
total enzyme concentration, ET, its maximum possible value.
• The initial velocity, v, then equals k2[ET]=Vmax
The Michaelis-Menten
Equation
• This equation states that the rate of an enzyme-catalyzed
reaction, v, at any moment is determined by two constants,
Km and Vmax, and the concentration of substrate at that
moment
• When S= Km , V= Vmax/2 : Km is the substrate concentration
that gives a velocity equal to one-half the maximal velocity64
• In the rectangular hyperbola, as [S] is increased, v approaches
the limiting value, Vmax, in an asymptotic fashion
• When [S]>>Km, then v=Vmax. That is, v is no longer dependent on
[S], so the reaction is obeying zero-order kinetics
• When [S]<<Km, then v≈(Vmax/Km)[S]. That is v approximately
follows a first-order rate equation, v=k’[S], where k’ =Vmax/Km
65
•
•
•
•
•
The Features of Vmax and Km
Km is a constant for a particular enzyme and substrate and is
independent of enzyme and substrate concentrations.
Vmax depends on enzyme concentration, and at saturating
substrate concentration, it is independent of substrate
concentration.
Km and Vmax may be influenced by pH, temperature and other
factors
When k-1 >> k2 (when the formation of product is the rate
limiting step), [ES] is assumed to be at equilibrium with [E] and
[S] ,i.e, ES is dissociating more often to yield E and S than to
yield product
Under this condition, Km is simplified to k-1 /k1 which in turn is
equal to the dissociation constant , Ks. Where these conditions
hold, Km does represent a measure of the affinity of the enzyme.
for its substrate in the ES complex. However, this scenario does
66
not apply for most enzymes
• When [S] >> Km, the characteristic property of the turnover
number for an enzyme can be used
• This number provides information on how many times the
enzyme performs its catalytic function per unit time, or how
many times it forms the ES complex and is regenerated (turned
over) by yielding product
• The rate-limiting step of the enzymatic reaction can give a
good indicator of the turnover number, and hence, the kinetic
efficiency
• Vmax=k2[ES]=k2[ET]
• k2 is denoted as kcat and gives the value of the turnover number
• Catalase has the highest turnover number known; each
molecule of this enzyme can degrade 40 million molecules of
H2O2 in one second. At the other end of the scale, lysozyme
requires 2 seconds to cleave a bond in its substrate
• Under physiological conditions, [S] is seldom saturating, and kcat
67
itself is not particularly informative
• The in vivo ratio of [S]/Km usually falls in the range of 0.01 to 1.0,
so active sites often are not filled with substrate
• A meaningful index of the efficiency of enzymes under these
conditions could be derived as follows:
• When S<<Km the concentration of free enzyme, [E], is
approximately equal to [ET], so that
• kcat/Km is an apparent second-order rate constant for the
reaction of E and S to form product
• Because Km is inversely proportional to the affinity of the
enzyme for its substrate and kcat is directly proportional to the
kinetic efficiency of the enzyme, kcat/Km provides an index of the
catalytic efficiency of an enzyme operating at substrate
68
concentrations substantially below saturation amounts
• The ratio kcat/Km can be expressed as
• But k1 must always be greater than or equal to k1k2/(k-1+k2). That
is, the reaction can go no faster than the rate at which E and S
come together
• Thus, k1 sets the upper limit for kcat/Km. In other words, the
catalytic efficiency of an enzyme cannot exceed the diffusioncontrolled rate of combination of E and S to form ES
• In water , the rate constant for such diffusion is approximately
109/M.sec
• Those enzymes that are most efficient in their catalysis have
kcat/Km ratios approaching this value. Their catalytic velocity is
limited only by the rate at which they encounter S; enzymes this
efficient have achieved so-called catalytic perfection
• All E and S encounters lead to reaction because such enzymes
can channel S to the active site, regardless of where S hits E 69
•
•
•
•
Linear Plots for the Michaelis Menten Equation
Vmax can be approximated experimentally from a substrate
saturation curve and Km can be derived from Vmax/2, so the two
constants of the Michaelis–Menten equation can be obtained
from plots of v versus [S]
However, straight-line plots are easier to evaluate than curves,
and the Michaelis Menten equation is reformulated to yield
straight-line plots
The best known of these reformulations is the Lineweaver–Burk
double-reciprocal plot
Taking the reciprocal of both sides of the MM equation gives
• This conforms to y=mx+b (the equation for a straight line),
where y=1/v; m, the slope, is Km/Vmax; x=1/[S]; and b=1/Vmax.
The x-intercept of the line is -1/Km and y-intercept is 1/Vmax 70
The Effect of pH on Enzymatic Activity
• The pH-enzyme activity profile of most enzymes can be
depicted by a bell-shaped curve, exhibiting an optimal pH at
which activity is maximal
• The optimal pH is usually the same as the pH of the fluid in
which the enzyme functions. Thus, most enzymes in the body
have their highest activity between pH 6 and pH 8 (the pH of
71
human blood is about 7.4)
• However, pepsin, which must function at the low pH of gastric
juice, has maximal activity at about pH 2
• An enzyme possesses many ionizable side chains and prosthetic
groups that not only determine its secondary and tertiary structure
but that are actively involved in its active site
• Further, the substrate itself often has ionizing groups, and one or
another of the ionic forms may preferentially interact with the
enzyme. Changes in pH affect the binding of the substrate at the
active site of the enzyme and also the rate of breakdown of the
enzyme-substrate complex
72
•
•
•
•
•
The Effect of Temperature on Enzymatic Activity
Raising the temperature increases the rate of both uncatalyzed
and enzyme-catalyzed reactions by increasing the kinetic
energy and the collision frequency of the reacting molecules
Most enzymatic reactions double in rate for every 10°C rise in
temperature
However, heat energy can also increase the kinetic energy of
the enzyme to a point that exceeds the energy barrier for
disrupting the non-covalent interactions that maintain its
three-dimensional structure
The polypeptide chain then begins to unfold, or denature, with
an accompanying loss of catalytic activity
The temperature range over which an enzyme maintains a
stable, catalytically active conformation depends upon—and
typically moderately exceeds—the normal temperature of the
cells in which it resides
73
• Enzymes from humans generally exhibit stability at
temperatures up to 45–55°C
• By contrast, enzymes from the thermophilic microorganisms
that reside in volcanic hot springs or undersea hydrothermal
vents may be stable up to or even above 100°C
• For mammals and other homeothermic organisms, changes in
enzyme reaction rates with temperature assume physiologic
importance only in circumstances such as fever or hypothermia
74
Reactions of Two or More Substrates
• Enzymes frequently catalyze the reaction of two, three or even
more different molecules to give one, two, three or more
products
• Sometimes all of the substrate molecules must be bound to an
active site at the same time and are presumably lined up on the
enzyme molecule in such a way that they can react in proper
sequence
• In other cases, the enzyme may transform molecule A to a
product, and then cause the product to react with molecule B.
The number of variations is enormous
• Two common types of two-substrate, two-product reactions
(termed “Bi-Bi” reactions) are sequential and ping pong
reactions
Sequential or Single-Displacement Reactions
• In sequential reactions, both substrates must combine with the
75
enzyme to form a ternary complex before catalysis can proceed
• Sequential reactions are sometimes referred to as singledisplacement reactions because the group undergoing transfer is
passed directly, in a single step, from one substrate to the other
• Sequential reactions can be of two distinct types:
a. random, where either A or B may bind to the enzyme first,
followed by the other substrate
Random
b. ordered, where A, designated the leading substrate, must
bind to E first before B can be bound
One explanation for an ordered mechanism is that the
addition of A induces a conformational change in the
enzyme that aligns residues that recognize and bind B 76
Ordered
Ping Pong Reactions
• The term “ping-pong” applies to mechanisms in which one or
more products are released from the enzyme before all the
substrates have been added
• Ping-pong reactions involve covalent catalysis and a transient,
modified form of the enzyme
• Ping-pong Bi-Bi reactions are double displacement reactions.
The group undergoing transfer is first displaced from substrate
A by the enzyme to form product P and a modified form of the
enzyme (F). The subsequent group transfer from F to the
second substrate B, forming product Q and regenerating E, 77
constitutes the second displacement
Ping Pong
Enzyme Inhibition
• Enzyme inhibition is one of the ways by which enzyme
activity is regulated experimentally or naturally
• Several therapeutic drugs function by inhibition of a specific
enzyme (of humans or pathogens)
• Inhibitor studies have contributed much of the available
information about enzyme kinetics and mechanisms
• Reversible inhibitors interact with an enzyme through noncovalent association/dissociation reactions
• In contrast, irreversible inhibitors usually cause stable,
covalent alterations in the enzyme
78
• Reversible inhibition is further divided into three types:
1. Competitive Inhibition
• In this type of reversible inhibition, a compound competes
with an enzyme’s substrate for binding to the active site,
• This results in an apparent increase in the enzyme–substrate
dissociation constant (Ks ) (i.e., an apparent decrease in the
affinity of enzyme for substrate) without affecting the
enzyme’s maximum velocity (Vmax)
79
• The rate equation for the formation of product, the dissociation
constants for enzyme–substrate (ES) and enzyme–inhibitor (EI)
complexes and the enzyme mass balance are, respectively:
• Normalization of the rate equation by total enzyme
concentration (v/[ET ]) and rearrangement results in the
following expression for the velocity of an enzymatic reaction in
the presence of a competitive inhibitor:
80
• Ks∗ corresponds to the apparent enzyme–substrate
dissociation constant in the presence of an inhibitor. In the
case of competitive inhibition, Ks∗ =αKs
• Competitive inhibition can be relieved by increasing the
concentration of substrate
• Examples of competitive inhibition:
A classic example is the inhibition of succinate dehydrogenase
by succinate’s analogues malonate, oxalate or oxaloacetate
81
p-aminobenzoic acid is required by bacteria for the synthesis
of folic acid which functions as a coenzyme in one-carbon
transfer reactions that are important in amino acid
metabolism, in the synthesis of RNA and DNA and thus in cell
growth and division. Sulfanilamides are analogues of PABA
that inhibit the synthesis of folic acid
2. Uncompetitive Inhibition
• In this type of reversible inhibition, a compound interacts with
the enzyme– substrate complex at a site other than the active
site
• This results in an apparent decrease in both Vmax and Ks
82
• The apparent increase in affinity of enzyme for substrate (i.e., a
decrease in Ks ) is due to unproductive substrate binding,
resulting in a decrease in free enzyme concentration
• Half-maximum velocity, or half-maximal saturation, will
therefore be attained at a relatively lower substrate
concentration
• The rate equation for the formation of product, the dissociation
constants for enzyme–substrate (ES) and ES–inhibitor (ESI)
complexes and the enzyme mass balance are, respectively 83
• Normalization of the rate equation by total enzyme
concentration (v/[ET ]) and rearrangement results in the
following expression:
• Vmax∗ and Ks∗ correspond, respectively, to the apparent enzyme
maximum velocity and apparent enzyme–substrate dissociation
constant in the presence of an inhibitor
• In the case of uncompetitive inhibition, Vmax∗= Vmax/α and
84
Ks∗= Ks/α
3. Mixed Inhibition
• In this type of reversible inhibition, a compound can interact
with both the free enzyme and the enzyme–substrate complex
at a site other than the active site:
• This results in an apparent decrease in Vmax and an apparent
increase in Ks
• The rate equation for the formation of product, the dissociation
constants for enzyme–substrate (ES and ESI) and enzyme–
inhibitor (EI and ESI) complexes, and the enzyme mass balance
85
are, respectively,
• Normalization of the rate equation by total enzyme
concentration (v/[ET ]) and rearrangement results in the
following expression:
• In the case of mixed inhibition, Vmax∗= Vmax/β and Ks∗=(α/ β)Ks
86
Non-Competitive Inhibition is a special case of mixed inhibition
where δ = 1 and α = β. Thus, the expression for the velocity of an
enzymatic reaction in the presence of a non-competitive inhibitor
becomes:
Competitive Inhibition
• Thus, for non-competitive inhibition, an apparent decrease in Vmax
is observed while Ks remains unaffected
A Comparison of the Effects of Different Types of Inhibitors
87
Mixed Inhibition
• In the cases of uncompetitive
and mixed inhibition, the
activity of the enzyme is fully
restored when the inhibitor is
removed from the system (by
dialysis, gel filtration or other
separation techniques) in
which the enzyme functions
Irreversible Inhibition
• Irreversible inhibition occurs when the inhibitor reacts at or
near the active site of the enzyme with covalent
modification of the active site or when the inhibitor binds
so tightly that, there is no dissociation of enzyme and
inhibitor
• Thus, physical separation processes are ineffective in
88
removing the irreversible inhibitor from the enzyme
• The serine protease family comprises of enzymes such as
chymotrypsin, trypsin, acetylcholinesterase and thrombin;
acetylcholinesterase shows a similar mechanism and is a serine
esterase
• The essential serine residues in these enzymes can be inhibited
by different agents resulting in inactivation
• The lethal compound diisopropyl phosphofluoridate (DIFP) is an
organophosphorus compound that served as a prototype for the
development of the nerve gas sarin and other organophosphorus toxins, such as the insecticides malathion and
parathion
• DIFP exerts its toxic effect by forming a covalent intermediate in
the active site of acetylcholinesterase, thereby preventing the
enzyme from degrading the neurotransmitter acetylcholine
• Once the covalent bond is formed, the inhibition by DIFP is
essentially irreversible, and activity can only be recovered as new
89
enzyme is synthesized
90
• Aspirin (acetylsalicylic acid) provides an example of a
pharmacologic drug that exerts its effect through the covalent
acetylation of an active site serine in the enzyme prostaglandin
endoperoxide synthase (cycloxygenase)
• Aspirin resembles a portion of the prostaglandin precursor that
is a physiologic substrate for the enzyme
91
• Mechanism-based inhibitors are a group made up of irreversible
inhibitors and transition state analogs
• Transition state analogs are extremely potent and specific
inhibitors of enzymes because they bind so much more tightly to
the enzyme than do substrates or products
• Drugs cannot be designed that precisely mimic the transition
state because of its highly unstable structure
• However, substrates undergo progressive changes in their
overall electrostatic structure during the formation of a
transition state complex, and effective drugs often resemble an
intermediate stage of the reaction more closely than they
resemble the substrate
• Such compounds are often referred to as substrate analogs,
even though they bind more tightly than substrates
• The antibiotic penicillin is a transition state analog that binds
very tightly to glycopeptidyl transferase, an enzyme required by
92
bacteria for synthesis of the cell wall
• Glycopeptidyl transferase catalyzes a partial reaction with penicillin
that covalently attaches penicillin to its own active site serine
• The reaction is favored by the strong resemblance between the
peptide bond in the β-lactam ring of penicillin and the transition
state complex of the natural transpeptidation reaction
• Active site inhibitors such as penicillin that undergo partial
reaction to form irreversible inhibitors in the active site are
sometimes termed “suicide inhibitors” or “Trojan horse
substrates”
93
•
•
•
•
•
•
•
Enzyme Regulation
The metabolic rate of key substances, which can proceed in
multiple pathways, is regulated and integrated
This regulation and integration is the result of the control
exerted over the activity of enzymes
A metabolic pathway involves many enzymes functioning in an
ordered manner to carry out a particular metabolic process
Control of a pathway is accomplished through modulation of
the activity of only one or a few key enzymes
These regulatory enzymes usually catalyze the first or an early
reaction in a metabolic sequence
A regulatory enzyme catalyzes a rate-limiting (or ratedetermining) chemical reaction that controls the overall
pathway. It may also catalyze a chemical reaction unique to
that pathway, which is known as a committed step
The rate-limiting step need not be the same as the committed
94
step
• Those enzymes which catalyze the rate-limiting step or the
committed step of a pathway are under regulation
• When the a product exceeds the steady-state level
concentration, it inhibits the regulatory enzyme in an
attempt to normalize the overall process. This type of control
is known as feedback inhibition
• Regulation can be based upon two main aspects of enzymes
–their amounts and/or activities
Control Over Amount
• There are genetic controls over the amounts of enzyme
synthesized by cells
• If the gene encoding a particular enzyme protein is turned on
or off, changes in the amount of enzyme activity soon follow
• Induction, which is the activation of enzyme synthesis, and
repression, which is the shutdown of enzyme synthesis, are
important mechanisms for the regulation of metabolism 95
• By controlling the amount of an enzyme that is present at
any moment, cells can either activate or terminate various
metabolic routes
• Genetic controls over enzyme levels have a response time
ranging from minutes in rapidly dividing bacteria to hours (or
longer) in higher eukaryotes
• In addition to the regulation of the rate of synthesis of
enzymes, the degradation of enzymes induced by physical
and/or chemical changes in the enzyme, may determine the
amount of enzyme present
Control Over Catalytic Activity
• Changes in intrinsic catalytic efficiency effected by binding of
dissociable ligands (allosteric regulation), covalent
modification, proteolytic cleavage or protein-protein
interaction achieve regulation of enzymatic activity within
96
seconds
• Changes in protein level serve long-term adaptive
requirements, whereas changes in catalytic efficiency are
best suited for rapid and transient alterations in metabolite
flux
Allosteric Regulation
• Those enzymes in metabolic pathways whose activities can
be regulated by non-covalent interactions of certain
compounds at sites other than the catalytic are known as
allosteric enzymes
• The term "allosteric" is of Greek origin, the root word
"allos" meaning "other’’
• Thus, an allosteric site is a unique region of an enzyme
other than the substrate binding site that leads to catalysis
• At the allosteric site, the enzyme is regulated by noncovalent interaction with specific ligands known as
97
effectors or modulators
• Allosteric enzymes are characterized by cooperativity: the
binding of a ligand to an allosteric site affects binding of the
substrate to the enzyme
• Binding of an allosteric modulator causes a change in the
conformation of the enzyme that leads to a change in the
binding affinity of the enzyme for the substrate
Allosteric enzymes are those having “other shapes” or
conformations induced by the binding of modulators
• The catalytically more active conformation of allosteric
enzymes is known as the relaxed or R-state or R-state; the less
active conformation is known as the taut or T-state
• The effect of a modulator may be positive (activatory) or
negative (inhibitory). The former leads to increased affinity of
the enzyme for its substrate, whereas the reverse is true for the
latter. Activatory sites and inhibitory sites are separate and
specific for their respective modulators
98
• Feedback inhibitors are negative allosteric regulators
Subunits Interactions in Allosteric Enzymes
99
• Two types of interaction occur in allosteric enzymes:
homotropic and heterotropic
• In a homotropic interaction, the same ligand influences
positively the cooperativity between different modulator sites.
Heterotropic interaction refers to the effect of one ligand on the
binding of a different ligand
• Allosteric modulators should not be confused with
uncompetitive and mixed inhibitors
• Although the latter bind at a second site on the enzyme, they
do not necessarily mediate conformational changes between
active and inactive forms, and the kinetic effects are distinct
• Allosteric enzymes are generally larger and more complex than
non-allosteric enzymes. Most have two or more subunits
• For example, aspartate transcarbamoylase (ATCase), which
catalyzes an early reaction in the biosynthesis of pyrimidine
nucleotides, has 12 polypeptide chains organized into catalytic
100
and regulatory subunits
• Allosteric enzymes show relationships between V and [S] that
differ from Michaelis-Menten kinetics
• For homotropic allosteric enzymes, plots of V versus [S]
produce a sigmoid saturation curve, rather than the hyperbolic
curve typical of non-regulatory enzymes
• Sigmoid kinetic behavior generally reflects cooperative
interactions between protein subunits
• Changes in the structure of one subunit are translated into
structural changes in adjacent subunits, an effect mediated by
non-covalent interactions at the interface between subunits
• On the sigmoid saturation curve the value of [S] at which V is
half-maximal is referred to as K0.5(instead of Km)
• For heterotropic allosteric enzymes, it is difficult to generalize
about the shape of the substrate-saturation curve
• An activator may cause the curve to become more nearly
hyperbolic, with a decrease in K0.5 but no change in Vmax 101
• Other heterotropic allosteric enzymes respond to an activator
by an increase in Vmax with little change in K0.5
• A negative modulator may produce a more sigmoid substratesaturation curve, with increase in K0.5 or decrease in Vmax
The Sigmoid Curve of a Homotropic Enzyme
102
The Activity Curves of Heterotropic Allosteric Enzymes
103
•
•
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•
•
•
Covalent Modification
In another important class of regulatory enzymes, activity is
modulated by covalent modification of the enzyme molecule
Modifying groups include phosphoryl, adenylyl, uridylyl,
methyl and adenosine diphosphate ribosyl groups
Phosphorylation is the most common type of regulatory
modification; one-third to one-half of all proteins in a
eukaryotic cell are phosphorylated
Some proteins have only one phosphorylated residue, others
have several and a few have dozens of sites for
phosphorylation
The attachment of phosphoryl groups to specific amino acid
residues of a protein is catalyzed by protein kinases; removal
of phosphoryl groups is catalyzed by phosphoprotein
phosphatases
Phosphoprotein phosphatases show less substrate specificity
104
than protein kinases
• The addition of a phosphoryl group to a Ser, Thr or Tyr residue
introduces a bulky, charged group into a region that was only
moderately polar
• The oxygen atoms of a phosphoryl group can hydrogen-bond
with one or several groups in a protein, commonly the amide
groups of the peptide backbone or an arginine residue
• The two negative charges on a phosphorylated side chain can
also repel neighboring negatively charged (Asp or Glu) residues
• When the modified side chain is located in a region of the
protein critical to its three dimensional structure,
phosphorylation can have dramatic effects on protein
conformation and thus on substrate binding and catalysis
105
106
Examples of Covalent Modification
•
•
•
•
•
Proteolytic Activation (and Protein-Protein Interaction)
Most proteins become fully active as their synthesis is
completed and they spontaneously fold into their native, threedimensional conformations
Some proteins,however, are synthesized as inactive precursors,
called zymogens or proenzymes, that only acquire full activity
upon specific proteolytic cleavage of one or several of their
peptide bonds
Unlike allosteric regulation or covalent modification, zymogen
activation by specific proteolysis is an irreversible process
The synthesis of zymogens as inactive precursors prevents
them from cleaving proteins prematurely at their sites of
synthesis or secretion
Chymotrypsinogen, for example, is stored in vesicles within
pancreatic cells until secreted into ducts leading to the
intestinal lumen. In the digestive tract, chymotrypsinogen is
107
converted to chymotrypsin by the proteolytic enzyme trypsin
• Trypsin cleaves off a small peptide from the N-terminal
region (and two internal peptides)
• This cleavage activates chymotrypsin by causing a
conformational change in the spacing of amino acid residues
around the binding site for the denatured protein substrate
and around the catalytic site
• Most of the proteases involved in blood clotting are
zymogens, such as fibrinogen and prothrombin, which
circulate in blood in the inactive form
• They are cleaved to the active form (fibrin and thrombin,
respectively) by other proteases, which have been activated
by their attachment to the site of injury in a blood vessel wall
• Thus, clots form at the site of injury and not randomly in
circulation
• Because proteolytic activation is irreversible, a way of
inactivating enzymes is needed
108
109
• Most proteolytic enzymes of the digestive and the blood
coagulation system contain serine in the active centre and are
inhibited by the binding of serine protease inhibitors (serpins)
• There are more than 40 serpins in blood and they play an
important role in inhibition of some powerful proteolytic
enzymes, e.g. those involved in blood clotting
• One of the best-known examples of a serpin is α1antiproteinase (antitrypsin), which inhibits elastase. Elastase is
a proteolytic enzyme that is secreted by macrophages, among
other cells
• Elastase is one of the many weapons used by macrophages to
kill invading pathogens. The macrophages are particularly
important in the lung, since it is a relatively easy point of entry
for pathogens
• However, once the pathogen has been killed, elastase activity
must be inhibited and this is achieved by release of α1110
antitrypsin
• The free radicals in tobacco smoke cause chronic damage to the
cells of the lung; they also decrease the affinity of α1antiproteinase for elastase. The resultant damage to the lung
tissue usually leads to emphysema
• Another example of serpins is, pancreatic trypsin inhibitor,
which binds to and inhibits trypsin
• In contrast with nearly all known protein assemblies, the
trypsin-pancreatic trypsin inhibitor complex is not dissociated
into its constituent chains by treatment with denaturing agents
such as 8 M urea or 6 M guanidine hydrochloride
o Some protein hormones are synthesized in the form of inactive
precursor molecules, from which the active hormone is derived by
proteolysis. For instance, insulin is generated by proteolytic
excision of a specific peptide from proinsulin
o Collagen also is initially synthesized as the soluble precursor
111
procollagen
•
•
•
•
Clinical Applications of Enzymes
When a tissue is damaged, infected or inflamed, cell
membranes become more permeable or are destroyed so that
the content of the cytoplasm, especially the dissolved
substances, are able to pass to the extracellular space and then
into the bloodstream
The blood can be drawn, plasma can be prepared and the
enzymatic activity can be measured
These measurements have been useful, particularly in the
diagnosis of damage to the heart, liver and muscle, and enzyme
measurements are useful in following the healing process and
the prognosis
Plasma-specific enzymes are enzymes that are normally
present in plasma, perform their primary function in blood and
have levels of activity that are usually higher in plasma than in
tissue cells. Examples are enzymes involved in blood clotting
112
and immune response
• Non-plasma-specific enzymes are intracellular enzymes
normally present in plasma at minimal levels or at
concentrations well below those in tissue cells
• Their presence in plasma is normally due to turnover of tissue
cells, but they are released into the body fluids in excessive
concentrations as a result of cellular damage or impairment of
membrane function
• Normal cell turnover is carried out through a process known
as apoptosis (from the Greek for “shedding’’)
• Apoptosis is a programmed cell death which may be initiated
when cells fail to receive a life-maintaining signal (suicide) or
when they receive a death signal (murder)
• Cells dying by apoptosis undergo characteristic morphological
changes: they shrink and condense; the cytoskeleton
collapses; the nuclear envelope disassembles; and the nuclear
chromatin condenses and breaks into fragments
113
• The cell surface often blebs and breaks up into membraneenclosed fragments called apoptotic bodies
• The apoptotic bodies are engulfed by neighboring cells or
macrophages before they can spill their contents
• In this way, the cell dies neatly and is rapidly cleared away,
without causing a damaging inflammatory response
• In contrast to apoptosis, cells that die accidentally, in
response to an acute insult, usually do so by a process
called cell necrosis (from Greek for “corpse”)
• Necrotic cells swell and burst, spilling their contents over
their neighbors and eliciting an inflammatory response
• The enzymes that are measured in clinical laboratories are
those that are released from necrotic cells
• Certain enzymes have been of interest over the years
114
115
• The enzymes concentrated in the heart and liver are aspartate
aminotransferase (also called serum glutamate-oxaloacetate
transaminase (SGOT)) and alanine aminotransferase (also
•
•
•
•
•
called serum glutamate-pyruvate transaminase (SGPT))
Alkaline phosphatase (AP) is reflective of bone, intestine
and other tissues. Creatine kinase (CK) is reflective of
skeletal and cardiac muscle
Lactate dehydrogenase (LDH) is reflective of heart, liver,
muscle and red blood cells
α- amylase reflects the pancreas and acid phosphatase
reflects the prostate gland
By measuring different enzyme activities, a pattern is seen
that characterizes an organ
However, the search can be made more specific and
defining when the isoenzymes/isozymes, if present, are
116
measured
• Isozymes are quaternary forms of an enzyme differing in
their relative proportions of structurally equivalent but
catalytically distinct polypeptide subunits
• A classic example of isozymes is mammalian LDH which
exists as five different isozymes, depending on the
tetrameric association of two different subunits, H (hearttype) and M (skeletal-muscle-type): H4, H3M, H2M2, HM3 and
M4
• The kinetic properties of the various LDH isozymes differ in
terms of their relative affinities for the various substrates
and their sensitivity to inhibition by product
• Another example could be CK, which is a dimer made up of
M (muscle-type) subunit and B (brain-type) subunit
• Consequently, there are three CK isozymes: MM, MB and BB
• Different tissues express different isozyme forms, as
117
appropriate to their particular metabolic needs
118