Download CHAPTER 11 Mechanism of Enzyme Action

CHAPTER 11
Mechanism of Enzyme Action
1. General properties of enzymes
2. Activation energy and the
reaction coordinate
3. Catalytic mechanism
4. Lysozyme
5. Serine proteases
Enzyme act with great
speed and precision
Introduction
1.  Enormous variety of chemical reactions within a cell
2.  Mediated by Enzymes
3.  Enzymology, the study of enzymes
(coined 1878; Greek: en, in; zyme, yeast),
fermentation: glucose -> ethanol
12 enzyme-catalyzed steps
4.  James Summer, 1926, crystallized urease
from jack bean, shown to be a protein
5.  Other catalysts, i.e. ribozymes (peptide-bond
formation; “RNA-world”), only for units
6.  Proteins more versatile, 20 functional units
Introduction
Enzymes increase the rate of chemical
reactions by lowering the free energy
barrier that separates the reactants
and products
1.  General Properties of Enzymes
Enzymes differ from ordinary chemical
catalysts by:
-  Higher reaction rates, 106-1012
-  Milder reaction conditions (temp, pH, …)
-  Greater reaction specificity (no side products)
-  Capacity for regulation
Definition catalyst: catalyzes reaction but
is not itself consumed during the process
Table 11-1
A) Classification of Enzymes
-  naming: -ase, urease, alcohol dehydrogenase
but no rules,
-  systematic: IUBMB: 6 Classes acc. to the nature of
the chemical reaction that is catalyzed
(http://expasy.org/enzyme/)
B) Enzymes Act on Specific Substrates
-  Noncovalent forces through which substrates bind
to enzymes: van der Waals, electrostatic,
hydrogen bonding, hydrophobic intercations
-  Geometric Complementarity
-  Electronic Complementarity
-  Induced fit upon substrate binding
-  “lock-and-key” model (proposed by Emil Fischer)
An Enzyme-Substrate complex
Geometric and
electrostatic
complementarity
Enzymes are Stereospecific
-  Enzymes are highly specific both in binding to
chiral substrates and in catalyzing stereo-specific
reactions
-  Enzymes are themselves are chiral, L-amino acids
-> active centers = active site is asymmetric/
stereo selective
Citrate is prochiral and is stereospecifically transformed into isocitrate
Stereospecificity in
substrate binding
Enzymes vary in geometric Specificity
-  Stereoselectivity, right hand into left glove
-  Geometric specificity is a more stringent
requirement than stereoselectivity, old key into
modern lock:
i.e. alcohol dehydrogenase, oxidation of
ethanol (CH3CH2OH) to acetaldehyde (CH3CHO)
faster than methanol to formaldehyde
or isopropanol to aceton
even though they only differ by deletion or
addition of one CH2 group !
Some enzymes are very permissive,
chymotrypsin, can hydrolyze amide and
ester bonds, exception rather than rule !
Some Enzymes Require Cofactors
-  Can act as enzymes *chemical teeth” to take
over chemical reactions that cannot be performed
by amino acid side chains…
-  Required in diet of organisms
-  for example metal ions, Cu2+, Fe3+, Zn2+
toxicity, Cd2+ and Hg2+ can replace Zn and
inactivate the enzyme
-  organic molecules, coenzymes,
can transiently associate with enzyme as cosubstrate,
i.e., nicotinamide adenine dinucleotide (NAD+)
Types of Cofactors in Enzymes
The structure and reaction of NAD+
NAD+ is an obligatory cofactor in
The alcohol dehydrogenase (ADH) reaction
NADH dissociates from the enzyme
to be re-oxidized in an independent reaction
Prosthetic groups
Permanently associated with enzyme, often by
covalent bonds, example heme is bound to proteins
called cytochromes
Holoenzyme = enzyme+cofactor complex, active
Apoenzyme, lacks cofactor, inactive
Coenzymes must be regenerated
In order to complete the catalytic cycle, the
coenzyme must return to its original state
i.e. by a different enzyme such as is the case with
NADH
2) Activation Energy and the
Reaction Coordinate
Transition State Theory: developed in 1930s
HA-HB + HC -> HA + HB-HC
Transition state: HA--HB—HC
Transition state = point of highest free energy
= most unstable
Reactants approach one another along a path of
minimal free energy = reaction coordinate
Transition state diagram/reaction coordinate diagram:
Plot of free energy versus the reaction coordinate
Transition State Diagram
(Symetrical)
Transition
State
Substrate
Product
Transition State Diagram
(Asymetrical)
Free energy
of activation
Free energy
of reaction
Activation Energy and the
Reaction Coordinate
The greater the free energy of activation,
the slower the reaction rate
If the free energy of the reaction, ∆G<0,
then the reaction is spontaneous and
releases energy (heat)
Transition State Diagram
For a Two-Step Reaction
Rate-determining
“bottleneck”
Catalysts Reduce the free energy of
activation, ∆G‡
Catalysts act by providing a reaction
pathway with a transition state whose free
energy is lower than that of the uncatalyzed reaction
Effect of a catalyst on the transition
state diagram of a reaction
Catalysts Reduce the free energy of
activation, ∆G‡
Reaction rate is proportional to e-∆G‡/RT
∆∆G‡ of 5.7kJ/mol (1/2 of one hydrogen
bond) gives 10-fold rate enhancement
∆∆G‡ of 34kJ/mol (small fraction of a
covalent bond) give 106-fold enhancement
Note: the catalyst enhances rate of
forward and that of the back reaction by
the same magnitude, but ∆Greaction
determines whether forward or back reaction
is favored
3) Catalytic Mechanisms
Enzymes lower the free energy of the
transition state (∆G‡) by stabilizing the
transition state
Learn about enzymatic reactions mechanisms
by examining the corresponding non-enzymatic
reactions of model compounds
Catalytic Mechanisms
Curved arrow convention to trace electron pairs
At all times, rules of chemical reasons apply to
the system, i.e. never five bonds on C, or 2 on H
etc.
Types of Catalytic Mechanisms
1.  Acid-base catalysis
2.  Covalent catalysis
3.  Metal ion catalysis
4.  Proximity and orientation effects
5.  Preferential binding of the transition state
A) Acid-Base Catalysis occurs
by Proton Transfer
General acid catalysis: Proton transfer from
an acid lowers the free energy of a
reaction’s transition state
Example, keto-enol tautomerization (a)
Enhanced by proton donation (b) or
proton abstraction (c) (general base
catalyzed)
Concerted Acid-Base Catalysis
Asp, Glu, His, Cys, Tyr, Lys have pK’s in or
near the physiological range
The ability of enzymes to arrange several
catalytic groups around their substrates
makes concerted acid-base catalysis a
common enzymatic mechanism
Effects of pH on Enzyme Activity
Most enzymes are active only within a narrow pH
range of 5-9.
Reaction rates exhibit bell-shaped curves in
dependence of pH (reflects ionization state of
important residues)
pH optimum gives information about catalytically
important residues, if 4/5 -> Glu, Asp; 6->His,
10->Lys
pK of residues can vary depending on chemical
environment +/- 2
pH Optimum of Fumarase
RNase A is an acid-base catalyst
Bovine pancreatic RNase A: Digestive enzyme
secreted by pancreas into the small intestine
2’,3’ cyclic nucleotides isolated as intermediates
pH-dependence indicates 2 important His, 12, 119
that act in a concerted manner as general acid
and base catalysts to catalyze a two-step
reaction
X-ray structure of bovine pancreatic RNase S
UpcA substrate in active site
The RNase A mechanism
B) Covalent Catalysis Usually Requires
a Nucleophile
Covalent Catalysis accelerates reaction rates
through the transient formation of a
catalyst-substrate covalent bond
Usually, nucleophilic group on enzyme attacks
an electrophilic group on the substrate
= nucleophilic catalysis
Example: decarboxylation of acetoacetate
Decarboxylation of acetoacetate
Three stages of Covalent Catalysis
1.  Nucleophilic attack of enzyme on substrate
2.  Withdrawal of electrons
3.  Elimination of catalysts by reversion of step 1
(not shown above).
Nucleophilicity of a substance is
related to its basicity:
Important aspect of covalent catalysis
The more stable the covalent bond formed,
the less easily it can be decomposed in the
final step of a reaction
Good covalent catalysis must be (i) highly nucleophile
and (ii) form a good leaving group. These are
imidazole and thiol groups, i.e. Lys, His and Cys,
Asp, Ser, some coenzymes (thiamine
pyrophosphate, pyridoxal phosphate)
C) Metal Ion Cofactors Act as Catalysts
1/3 of known enzymes require metal ions for
catalysis
Metalloenzymes contain tightly bound metal ion
(Fe2+, Fe3+, Cu2+, Mn2+, Co2+),
Na+, K+, or Ca2+ play structural rather than
catalytic roles
Mg2+, Zn2+ may be either structural or catalytic
Metal Ion Cofactors Act as Catalysts
Metal ions participate in the catalytic process:
1. By binding to substrate to orient them
properly for reaction
2. By mediating oxidation-reduction reactions
through reversible changes in the metal ions
oxidation state
3. By electrostatically stabilizing or shielding
negative charges
Often: Metal ion acts similar to a proton, or
polarizes water to generate OH-
The role of Zn2+ in carbonic anhydrase
CO2 + H2O <-> HCO3- + H+
Zn2+ polarizes water, which then attacks CO2
D) Catalysis can occur through proximity
and orientation effects
Enzymes are much more efficient catalysts than
organic model compounds
Due to proximity and orientation effects
Reactants come together with proper spatial
relationship
Example: p-nitrophenylacetate
intramolecular reaction is 24 times faster
Inter- versus intramolecular reaction
24-times
faster
Catalysis can occur through proximity
and orientation effects
Enzymes are usually much bigger than their
substrates
By oriented binding and immobilization of the
substrate, enzymes facilitate catalysis by four
ways
1. bring substrates close to catalytic residues
2. Binding of substrate in proper orientation (up
to 102-fold)
3. Stabilization of transition state by
electrostatic interactions
4. freezing out of translational and rotational
mobility of the substrate (up to 107-fold)
The geometry of an SN2 reaction
E) Enzymes catalyze reactions by
preferentially binding the transition state
An enzyme may binds the transition state of the
reaction with greater affinity than its substrate
or products
This together with the previously discussed factors
accounts for the high rate of catalysis
For example, if enzyme binds the transition state
with 34.2 kJ/mol (= 2 hydrogen bonds) it results
in 106-fold rate enhancement
315-times faster if R is CH3
rather than H
Effect of preferential transition
state binding
Transition state analogs are
enzyme inhibitors
For example proline racemase
Inhibitors
4) Lysozyme
Lysozyme is an enzyme that degrades bacterial cell
walls.
Hydrolyzes β(1->4) glycosidic bond from N-acetylmuramic
(NAM) acid to N-acetylglucosamine (NAG) in cell wall
peptidoglycan
also hydrolyzes chitin: β(1->4) NAG
Lysozyme occurs widely as bactericidal agent, best
characterized: hen egg white lysozyme, 14.3 kD, single
129 Aa polypeptide chain, 4 disulfide bonds, rate
enhancement 108-fold
The lysozyme cleavage site
β(1->4) Lysozyme’s catalytic site was
identified through model
Lysozyme structure solved by X-ray in 1965, first enzyme
Ellipsoidal shape with prominent cleft in substrate bdg site,
That traverse one face of the molecule
Use model building to understand enzyme substrate
interactions
6 saccharide units, A-F
In D ring, C6 and O6 too closely contact enzyme
=> distortion of glucose ring from chair => half chair
=> have to move from Lysozyme’s catalytic site was
identified through model
Chair and half-chair conformation
Distortion of D ring, Saccharide unit 4
=> C1, C2, C5 , and O5 are coplanar
Stabilization through H bridges and ionic interactions
The interactions of lysozyme
with its substrate
Identification of the bond
that lysozyme cleaves
D-ring remains
β anomer
B) The lysozyme reaction proceeds via
a covalent intermediate
The reaction catalyzed by lysozyme, the hydrolysis of a
glycoside, is the conversion of an acetal into a
hemiacetal
Non-enzymatic, this is an acid-catalyzed reaction,
involving the protonation of an oxygen atom, followed by
cleavage of a O-C bond -> transient formation of
resonance stabilized carbocation = oxonium ion
Enzymatic reaction should include an acid catalyst and a
stabilization of the oxonium ion transition state
The mechanism of the nonenzymatic acidcatalyzed hydrolysis of an acetal to a hemiacetal
Glu 35 and Asp 52 are lysozyme’s catalytic residues
Transition state analog inhibition of
lysozyme
NAG lactone binds to the D subside with about 9.2 kJ/
mol greater affinity than does NAG (corresponds to a 40fold enhancement)
Observation of the covalent intermediate
The lifetime of a glucosyl oxonium ion in water is ~10-12 sec
To be observed: its rate of formation must be greater
than that of its breakdown
1. Formation slowed by substituting F at C2 of D ring to draw electrons
2. Mutating Glu 35 to Gln (E35Q) to remove general acid base catalyst
3. Substitution F at C1 of D ring as good leaving group
4) Serine Proteases
Class of proteolytic enzymes,
Active site reactive Ser-residue (≠cut after Ser !)
digestive enzymes,
developmental regulation
blood clotting
inflammation
many other cellular processes
Focus on chymotrypsin, trypsin, elastase
A) Active site residues were identified
by chemical labeling
Chymotrypsin, trypsin, elastase are digestive enzymes
synthesized by the pancreas, secreted into duodenum
All cleave peptide bonds but with different specificities
for side chain residues
Chymotrypsin: after bulky hydrophobic residue
Trypsin: after positively charged residue
Elastase: after small neutral residue
Chemical labeling with diisopropylphosphofluoridate (DIPF)
Reacts only with Ser 195 of chymotrypsin, very toxic
Does not label other Ser, why ?
Diisopropylphosphofluoridate (DIPF)
Diisopropylphosphofluoridate (DIPF)
A second important residue, His 57, was identified
by affinity labeling
Substrate analog bearing reactive groups reacts with
nearby residues, “Trojan horses”
Chymotrypsin specifically binds tosyl-L-phenylalanine
chloromethylketone (TPCK), resembles Phe, reacts
with His 57
B) X-ray structures provide information bout
catalysis, substrate specificity, and evolution
Chymotrypsin, trypsin, elastase are strikingly
Similar
Have ca. 240 Aa, 40% identical
All have reactive Ser and important His
Closely related 3D structure, chymotrypsin solved
in 1967
Active site His 57, Ser 195, Asp 102 form
Catalytic triad residues
X-ray structure of bovine trypsin
in complex with leupeptin
The active site residues of chymotrypsin
Nerve Poisons
Use of DIPF as enzyme inhibitor based on discovery that
organophosphorous compounds, such as DIPF, acts as potent
nerve poisons.
Inactivate acetylcholinesterase, catalyzes hydrolysis of
acetylcholine, active site Ser
Nerve Poisons
Acetylcholine is a neurotransmitter: transmits nerve
impulses across certain types of synapses (junctions
between nerve cells)
Acetylcholinesterase in the synaptic clevt normally degrades
acetylcholine to terminate nerve impulse.
⇒ Acetylcholine receptor, which is a Na+-K+ channel, remains
open for longer than normal, toxic to humans (inability to
breathe)
DIPF so toxic that it has been used as military nerve gas.
Related compound such as parathion and malathion are
used as insecticides
Used by terrorists in Tokyo subway, 1995
Inactivated by paraoxonase, expressed at
different levels in different individuals, different
sensitivity to nerve toxins of this class
Tetrahedral phosphate
= transition state analog
Substrate specificities are only
partially rationalized
X-ray structure suggest the basis for the
Different substrate specificities of
chymostrypsin,trypsin and elastase
1. In chymotrypsin, preferred Phe,
Trp or Tyr fit into a slitlike
hydrophobic pocket located near
the catalytic groups
Specificity pockets of three serine proteases
2. In trypsin, the Ser
198 of chymotrypsin,
which lies at the
bottom of the binding
pocket is replaced by
Asp. Form ion pairs
with Arg and Lys in
substrate. But equally
deep slitlike pocket as
in chymotrypsin
But Asp->ser 189 mutation
does not convert Trypsin
into chymotrypsin
Specificity pockets of three serine proteases
3. In elastase, hydolyzes
the nearly indegstible
Ala, Gly, and Val-rich
protein elastin
(connective tissue)
Bdg pocket contains Val
and Thr instead of the
two Gly found in trypsin
and chymotrypsin ->
cleaves substrates with
small neutral side chains
Serine proteases exhibit divergent and
convergent evolution
Great overall similarity -> arose
through duplication of an ancestral
enzyme, followed by divergent
evolution of the resulting enzyme
Primordial enzyme arose before
separation of pro- and eukaryote
Other Ser-proteases, however,
have very little homology, i.e,
subtilisin and serine
carboxypeptidase II
Arose through convergent
evolution
C) Serine proteases use several
catalytic mechanisms
Catalytic mechanism of chymotrypsin, based on
structural and chemical data. Applies to all Ser
proteases and other hydrolytic enzymes (lipases….)
1. After chymotrypsin has bound substrate:
Ser 195 nucleophilic attack on peptide’s
carbonyl group to form tetrahedral intermediate,
resembles transition state of this covalent
catalysis,
Proton on Ser is abosrbed by His 57 to fomr
imidazolium ion (general base catalysis), aided by
Asp 102
Formation of the tetrahedral intermediate
2. Decomposition of the
tetrahedral intermediate
Decomposition to the acylenzyme intermediate and
scission of the peptide bond
Driven by donation of proton
from N3 of His 57 (general
acid catalysis)
Helped by polarizing effect
of Asp 102 on His 57
(electrostatic catalysis)
3. Amine leaving group is replaced by water
The amine leaving group (the new N-terminus of the
cleaved peptide) is released from the enzyme and
replaced by water from the solvent
4. Hydrolysis of the acylenzyme intermediate
By the addition of water,
formation of a second
tetrahedral intermediate
5. Reversal of step 1
Yields the carboxylate product, that is the new Cterminus of the peptide, and regenerates the
active enzyme
Serine proteases preferentially bind the
transition state
1.  Conformational distortion that occurs with formation of
the tetrahedral intermediate causes the anionic carbonyl
oxygen to move deeper into the active site so as to
occupy the oxyanion hole
2.  There it forms two hydrogen bonds with the enzyme
the oxyanion hole is conserved in chymotrypsin and
subtilisin, convergent evolution
3.  This tetrahedral distortion allows formation of another
hydrogen bond between Gly 193 and the backbone NH of
the residue preceding the scissile peptide bond
Transition state stabilization in the serine
proteases
Transition state stabilization in the serine
proteases
The preferential binding of the transition
state (or the tetrahedral intermediate)
over the enzyme-substrate complex or the
acyl-enzyme intermediate is responsible for
much of the catalytic efficiency of serine
proteases
Mutating any or all residues of the catalytic triad
yields enzymes that still enhance proteolysis by ca. 5
104-fold over the noncatalyzed reaction, native
enzyme 1010
Low-barrier hydrogen bonds may stabilize
the transition state
1.  Proton transfer between hydrogen donor and acceptor
occurs at reasonable rates only when the pK of the donor
is 2-3 pH units greater than that of the protonated form
of the acceptor
2.  If their pK values of proton donor and acceptor are
nearly equal, the distinction breaks down and: the
hydrogen atom becomes more or less equally shared
between them (D---H---A).
3.  Such low-barrier hydrogen bonds (LBHBs) are unusually
strong and short (40-80 kJ/mol versus 12-30 kJ/mol;
2.55-2.65Å versus 2.8-3.1Å)
4.  LBHBs don’t exist in aqueous phase but can form in the
environment of an enzyme
The tetrahedral intermediate resembles the
complex of trypsin with trypsin inhibitor
1. 
Strong evidence for formation of a tetrahedral intermediated
provided by X-ray structure of trypsin with bovine pancreatic trypsin
inhibitor (BPTI)
2. 
BPTI, 58 Aa, prevents self-digestion of organ of prematurely
activated trypsin, k= 1013 Mol, one of the strongest protein
interactions known
3. 
A Lys on BPTI occupies trypsin’s specificity pocket
4. 
But proteolytic reaction cannot proceed because the active site is so
tightly sealed that the leaving group does not dissociate and water
cannot enter
5. 
Protease inhibitors are common, e.g. plant defence against insects,
10% of blood plasma (a1-proteinase inhibitor against leukocyte
elastate (inflammation))
The tetrahedral intermediate resembles the
complex of trypsin with trypsin inhibitor
The tetrahedral intermediate has been
directly observed
Since the tetrahedral intermediate resembles the
transition state, it is thought to be unstable and
short-lived. Acly-enzyme complex is table at pH
5.0 (His 57 is protonated an cannot act as base
catalyst) and could be observed by X-ray
Immersing the acyl-enzyme crystals a pH 9 triggers
the hydrolytic reaction
Freeze crystals in liquid N2 and analyze by X-ray
Structure of the acyl-enzyme and
tetrahedral intermediates
D) Zymogens are inactive enzyme precursors
Proteolytic enzymes are usually made as larger,
inactive precursors = zymogens (proenzymes)
Acute pancreatitis is characterized by premature
activation of digestive enzymes
Enteropeptidase converts trypsinogen into trypsin,
Ser-protease under hormonal control, made in the
duodenal mucosa, cleaves lys 15 – Ile 16 = trypsin
cleavage site, i.e. self activation / autocatalytic
Also proelastase, procarboxypeptidase A, B, and
prophospholipase A2 are all activated by trypsin
The activation of trypsinogen to trypsin
Zymogens have distorted active sites
Liberation of N-terminal peptide results in
conformational change and activation of
the enzyme
The blood coagulation cascade
If blood vessel is damaged, clot forms as result of
platelet aggregation (small enucleated blood cells)
and formation of insoluble fibrin network that
traps additional blood cells
Fibrin is produced from the soluble circulating
fibrinogen through activation of the ser protease
thrombin
Thrombin is the last enzyme in a coagulation cascade
of enzymes, activation occurs on platelets
Initiated by membrane protein, tissue factor, forms
complex with circulating factor VII (extrinsic
pathway)
The blood coagulation cascade
The blood coagulation cascade
Intrinsic pathway activated by glass surface (negative
charge)
Congenital defects in factor VIII (hemophilia a) or factor
IX (hemophilia b)