Chapter 14
Mechanisms of Enzyme Action
Biochemistry
by
Reginald Garrett and Charles Grisham
Essential Question
Although the catalytic properties of enzymes may
seem almost magical, it is simply chemistry–
the breaking and making of bonds– that give
enzymes their prowess
 What are the universal chemical principles that
influence the mechanisms of these and other
enzymes
 How may we understand the many other
cases, in light of the knowledge gained from
these examples?
Outline of Chapter 14
1.
2.
3.
4.
5.
6.
7.
What Role Does Transition-State Stabilization Play in
Enzyme Catalysis?
What Are the Magnitudes of Enzyme-Induced Rate
Accelerations?
Why Is the Binding Energy of ES Crucial to Catalysis?
What Roles Do Entropy Loss and Destabilization of the ES
Complex Play?
How Tightly Do Transition-State Analogs Bind to the Active
Site?
What Are the Mechanisms of Catalysis?
What Can Be Learned from Typical Enzyme Mechanisms?
14.1 – What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?
H-O-H + Cl
Reactant
d
d
H-O H Cl
Transition state
• Transition state (10-13sec)
• Intermediate (10-13~10-3sec)
HO + HCl
Products
Figure 14.1
Enzymes catalyze reactions by lowering the activation energy. Here the free energy of activation
for (a) the uncatalyzed reaction, DGu‡, is larger than that for (b) the enzyme-catalyzed reaction,
DGe‡.
• Reaction rate acceleration by an enzyme
means that the energy barrier between ES and
EX‡ must be smaller than the barrier between S
and X‡ (DGe ‡ ＜DGu‡)
• This means that the enzyme must stabilize the
EX‡ transition state more than it stabilizes ES
→Enzymes bind the transition state structure more
tightly than the substrate
14.2 – What Are the Magnitudes of
Enzyme-Induced Rated Accelerations?
14.2 – What Are the Magnitudes of
Enzyme-Induced Rated Accelerations?
•
Mechanisms of catalysis:
•
1.
1.
1.
1.
1.
1.
Entropy loss in ES formation
Destabilization of ES
Covalent catalysis
General acid-base catalysis
Metal ion catalysis
Proximity and orientation
14.3 – Why Is the Binding Energy of
ES Crucial to Catalysis?
• The favorable interactions between the
substrate and amino acid residues on the
enzyme account for the intrinsic binding
energy, DGb
• The intrinsic binding energy ensures the
favorable formation of the ES complex, but
not too favorable!
intrinsic
binding
energy
Figure 14.2
The intrinsic binding energy of the enzyme-substrate (ES) complex (DGb ) is
compensated to some extent by entropy loss due to the binding of E and S (TDS)
and by destabilization of ES (DGd) by strain, distortion, desolvation , and similar
effects. If DGb were not compensated by TDS and DGd, the formation of ES would
follow the dashed line.
Figure 14.3
(a) Catalysis does not occur if the ES complex and the transition state for the reaction
are stabilized to equal extents. (b) Catalysis will occur if the transition state is stabilized
to a greater extent than the ES complex (right). Entropy loss and destabilization of the
ES complex DGd ensure that this will be the case.
14.3 – Why Is the Binding Energy of
ES Crucial to Catalysis?
• If uncompensated, it makes the activation
energy for the enzyme-catalyzed reaction
unnecessarily large and wastes some of the
catalytic power of the enzyme
14.4 – What Roles Do Entropy Loss
and Destabilization of the ES Complex
Play?
Raising the energy of ES raises the rate
For a given energy of EX‡, raising the
energy of ES will increase the catalyzed
rate
• This is accomplished by
1. loss of entropy due to formation of ES
2. destabilization of ES by
• structural strain & distortion
• desolvation
• electrostatic effects
•
Figure 14.4
Formation of the ES complex results in a loss of entropy. Prior to binding, E and S
are free to undergo translational and rotational motion. By comparison, the ES
complex is a more highly ordered, low-entropy complex.
Figure 14.5
Substrates typically lose
waters of hydration in the
formation of the ES complex.
Desolvation raises the
energy of the ES complex,
making it more reactive.
Figure 14.6
Electrostatic
destabilization of a
substrate may arise from
juxtaposition of like charges
in the active site. If such
charge repulsion is relieved
in the course of the reaction,
electrostatic destabilization
can result in a rate increase.
14.5 – How Tightly Do Transition-State
Analogs Bind to the Active Site?
• Transition state is exists only for about 10 -13 sec, less
than the time required for a bond vibration
• The nature of the elusive transition state can be explored
using transition state analogs
• Transition state analogs are stable molecules, chemically
and structurally similar to the transition state
• Transition-state analogs are only approximations of the
transition state itself and will never bind as tightly as
would be expected for the true transition state
Transition-State Analogs
Figure 14.7
The proline racemase reaction. Pyrrole-2-carboxylate and D -1-pyrroline-2carboxylate mimic the planar transition state of the reaction.
Figure 14.8
(a) Phosphoglycolohydroxamate
is an analog of the enediolate
transition state of the yeast
aldolase reaction. (b) Purine
riboside, a potent inhibitor of the
calf intestinal adenosine
deaminase reaction, binds to
adenosine deaminase as the 1,6hydrate. The hydrated form of
purine riboside is an analog of
the proposed transition state for
the reaction.
14.6 – What Are the Mechanisms of
Catalysis?
Covalent catalysis
• Some enzyme reactions derive much of their
rate acceleration from the formation of
covalent bonds between enzyme and substrate
BX + Y  BY + X
BX + Enz  E:B + X + Y  Enz + BY
• Most enzymes that carry out covalent catalysis
have ping-pong kinetic mechanisms
14.6 – What Are the Mechanisms of
Catalysis?
Covalent catalysis
• The side chains of amino acids in proteins offer a
variety of nucleophilic centers for catalysis,
including amines, carboxylate, aryl and alkyl
hydroxyls, imidazoles, and thiol groups
• These groups are readily attack electrophilic
centers of substrates, forming covalently bonded
enzyme-substrate intermediate
Figure 14.9
Examples of covalent bond
formation between enzyme
and substrate. In each case,
a nucleophilic center (X:) on
an enzyme attacks an
electrophilic center on a
substrate.
Glyceraldehyde-3-P + NAD+ + Pi  1,3-bisphosphoglycerate + NADH + H+
NAD+
NADH
Figure 14.10
Formation of a covalent intermediate in the glyceraldehyde-3-phosphate dehydrogenase
reaction. Nucleophilic attack by a cysteine —SH group forms a covalent acylcysteine
intermediate. Following hydride transfer to NAD+, nucleophilic attack by phosphate yields the
product, 1,3-bisphosphoglycerate.
14.6 – What Are the Mechanisms of
Catalysis?
General acid-base catalysis
• Specific acid-base catalysis involves H+ or
OH- that diffuses into the catalytic center
• General acid-base catalysis involves acids
and bases other than H+ and OH• These other acids and bases facilitate
transfer of H+ in the transition state
Figure 14.11
Specific and general acid - base
catalysis of simple reactions in
solution may be distinguished by
determining the dependence of
observed reaction rate constants
(kobs) on pH and buffer
concentration. (a) In specific acidbase catalysis, H+ or OHconcentration affects the reaction
rate, kobs is pH-dependent, but
buffers (which accept or donate
H+/OH-) have no effect. (b) In
general acid - base catalysis, in
which an ionizable buffer may
donate or accept a proton in the
transition state, kobs is dependent
on buffer concentration.
Figure 14.12
Catalysis of p-nitrophenylacetate hydrolysis by imidazole—an example of general base
catalysis. Proton transfer to imidazole in the transition state facilitates hydroxyl attack on
the substrate carbonyl carbon.
14.6 – What Are the Mechanisms of
Catalysis?
Low-barrier hydrogen bond (LBHB)
• The typical strength of a hydrogen bond is
10 to 30 kJ/mol
0.1 nm
1 order
0.18nm
0.07
0.25 nm (LBHB)
0.5 order
Low-Barrier Hydrogen Bonds
• As distance between heteroatoms becomes
smaller, H bonds become stronger
• Stabilization energies of LBHB may approach
60 kJ/mol in solution
• pKa values of the two electronegative atoms
must be similar
• Energy released in forming an LBHB can
assist catalysis
14.6 – What Are the Mechanisms of
Catalysis?
•
Metal ion catalysis
Many enzymes require metal ions for
maximal activity (metalloenzymes)
1. Stabilizing the increased electron
density or negative charge
2. Provide a powerful nucleophile at
neutral pH
M2+ + NucH
M2+(NucH)
M2+(NucH) + H+
14.6 – What Are the Mechanisms of
Catalysis?
Proximity
• Chemical reactions go faster when the reactants
are in proximity, that is, near each other
• Proximity and orientation play a role in enzyme
catalysis, but there is a problem with each of the
aforementioned comparisons
Figure 14.15
An example of proximity effects in catalysis. (a) The imidazole-catalyzed hydrolysis of pnitrophenylacetate is slow, but (b) the corresponding intramolecular reaction is 24-fold
faster (assuming [imidazole] = 1 M in [a]).
Figure 14.16
Orientation effects in intramolecular reactions can be dramatic. Steric crowding by methyl
groups provides a rate acceleration of 2.5 X 1011 for the lower reaction compared to the upper
reaction. (Adapted from Milstien,S., and Cohen, L.A., 1972. Stereopopulation control I. Rate enhancements
in the lactonization of o-hydroxyhydrocinnamic acid. Journal of the American Chemical Society 94:91589165.)
14.7 – What Can Be Learned from
Typical Enzyme Mechanisms?
• Serine proteases and aspartic proteases are
good examples
• Knowledge of the tertiary structure of an
enzyme is important
• Enzymes are the catalytic machines that sustain
life
The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA
• Serine proteases are a class of proteolytic
enzymes whose catalytic mechanism is based on
an active-site serine residue
• Ser is part of a "catalytic triad" of Ser, His, Asp
• Serine proteases are homologous, but locations of
the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
always as His-57, Asp-102, Ser-195
Figure 14.17
Comparison of the
amino acid sequences
of chymotrypsinogen,
trypsinogen, and
elastase. Each circle
represents one amino
acid. Numbering is
based on the sequence
of chymotrypsinogen.
Filled circles indicate
residues that are
identical in all three
proteins. Disulfide
bonds are indicated in
yellow. The positions of
the three catalytically
important active-site
residues (His57, Asp102,
and Ser195) are
indicated.
Serine Protease Mechanism
A mixture of covalent and general acid-base
catalysis
• Asp-102 functions only to orient His-57
• His-57 acts as a general acid and base
• Ser-195 forms a covalent bond with peptide
to be cleaved
• Covalent bond formation turns a trigonal C
into a tetrahedral C
• The tetrahedral oxyanion intermediate is
stabilized by N-Hs of Gly-193 and Ser-195
Figure 14.18
Structure of
chymotrypsin (white)
in a complex with eglin
C (blue ribbon
structure), a target
protein. The residues of
the catalytic triad (His57,
Asp102, and Ser195) are
highlighted. His57 (blue)
is flanked above by
Asp102 (red) and on the
right by Ser195 (yellow).
The catalytic site is
filled by a peptide
segment of eglin. Note
how close Ser195 is to
the peptide that would
be cleaved in a
chymotrypsin reaction.
Figure 14.19
The catalytic triad
of chymotrypsin .
Figure 14.20
The substrate-binding pockets of trypsin, chymotrypsin, and elastase. (Illustration:
Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without
permission. )
Serine Protease Mechanism
Kinetics
• The mechanism is based on studies of
the hydrolysis of artificial substrates–
simple organic ester
Figure 14.21
Artificial substrates used in studies of the mechanism of chymotrypsin .
Multistep mechanism
Figure 14.22
Burst kinetics observed in the chymotrypsin reaction. A burst of nitrophenolate production
is followed by a slower, steady-state release. After an initial lag period, acetate release is
also observed. This kinetic pattern is consistent with rapid formation of an acyl-enzyme
intermediate (and the burst of nitrophenolate ). The slower, steady-state release of
products corresponds to rate-limiting breakdown of the acyl-enzyme intermediate.
Serine Protease Mechanism
Kinetics
• In the chymotrypsin mechanism, the
nitrophenylacetate combines with the enzyme to
form an ES complex
• Followed by a rapid second step in which an
acyl-enzyme intermediate is formed, with the
acetyl group covalently bonded to the very
reactive Ser-195
Figure 14.23
Rapid formation of the acyl-enzyme intermediate is followed by slower product release.
Multistep mechanism
Figure 14.22
Burst kinetics observed in the chymotrypsin reaction. A burst of nitrophenolate production
is followed by a slower, steady-state release. After an initial lag period, acetate release is
also observed. This kinetic pattern is consistent with rapid formation of an acyl-enzyme
intermediate (and the burst of nitrophenolate ). The slower, steady-state release of
products corresponds to rate-limiting breakdown of the acyl-enzyme intermediate.
Figure 14.24
Diisopropylfluorophosphate (DIFP) reacts with active-site serine residues of serine proteases (and
esterases), causing permanent inactivation.
Figure 14.25
A detailed mechanism for the
chymotrypsin reaction. Note the
low-barrier hydrogen bond
(LBHB) in (c) and (g).
ES complex
Tetrahedral oxyanion
transition state
Acyl-enzyme intermediate
Acyl-enzyme-H2O
intermediate
Tetrahedral oxyanion
transition state
The Aspartic Proteases
•
•
•
•
Pepsin, chymosin, cathepsin D, renin and
HIV-1 protease
All involve two Asp residues at the active site
and two Asps work together as general acidbase catalysts
Active at acidic pH
Most aspartic proteases have a tertiary
structure consisting of two lobes (N-terminal
and C-terminal) with approximate two-fold
symmetry (fig 14.26)
HIV-1 protease is a homodimer
Figure 14.26
Structures of (a) HIV-1 protease, a dimer,
and (b) pepsin (a monomer). Pepsin’s Nterminal half is shown in red; C-terminal
half is shown in blue.
Figure 14.27
Acyl-enzyme and amino-enzyme intermediates originally proposed for aspartic proteases
were modeled after the acyl-enzyme intermediate of the serine proteases.
Aspartic Protease Mechanism
• pH dependence (fig 14.28)
• The aspartate carboxyl groups functioned
alternately as general acid and general base
• Deprotonated Asp acts as general base,
accepting a proton from HOH, forming OH- in
the transition state
• Other Asp (general acid) donates a proton,
facilitating formation of tetrahedral intermediate
Figure 14.28
pH-rate profiles for (a) pepsin and (b) HIV protease. (Adapted fro Denburg,J., et at., 1968. The
effect of PH on the rates of hydrolysis of three acylated dipeptiedes by pepsin.
Figure 14.29 A mechanism for the aspartic proteases. The letter titles describe the state as
follows: E represents the enzyme form with a low-barrier hydrogen bond between the catalytic
aspartates, F represents the enzyme form with one aspartate protonated and the other sharing in
the conventional hydrogen bond, S represents bound substrate, T represents a tetrahedral amide
hydrate intermediate, P represents bound carboxyl product, and Q represents bound amine
product. This mechanism is based in part on a mechanism proposed by Dexter Northrop, a
distant relative of John Northrop, who had first crystallized pepsin in 1930. The mechanism is also
based on data of Thomas Meek.
HIV-1 Protease
•
•
•
•
•
A novel aspartic protease
HIV-1 protease cleaves the polyprotein products
of the HIV genome, producing several proteins
necessary for viral growth and cellular infection
This is a remarkable imitation of mammalian
aspartic proteases
HIV-1 protease is a homodimer - more
genetically economical for the virus
Active site is two-fold symmetric
Two aspartate residues, Asp-25 and Asp-25’
Figure 14.31
HIV mRNA provides the genetic information for synthesis of a polyprotein . Proteolytic
cleavage of this polyprotein by HIV protease produces the individual proteins required for
viral growth and cellular infection.
Figure 14.32
(left) HIV-1 protease complexed with the inhibitor Crixivan (red) made by Merck.
The flaps (residues 46-55 from each subunit) covering the active site are shown in
green and the active site aspartate residues involved in catalysis are shown in
white. (right) The close-up of the active site shows the interaction of Crixivan with
the carboxyl groups of the essential aspartate residues.
Therapy for HIV?
•
•
•
•
Protease inhibitors as AIDS drugs
If the HIV-1 protease can be selectively
inhibited, then new HIV particles cannot form
Several novel protease inhibitors are currently
marketed as AIDS drugs
Many such inhibitors work in a culture dish
However, a successful drug must be able to kill
the virus in a human subject without blocking
other essential proteases in the body
Lysozyme
• Lysozyme hydrolyzes polysaccharide chains
and ruptures certain bacterial cells by
breaking down the cell wall
• Hen egg white enzyme has 129 residues with
four disulfide bonds (8 cysteine residues)
• Lysozyme hydrolyzes the glycosidic bond
between C-1 of NAM and C-4 of NAG
Figure 14.33
The lysozyme reaction.
Figure 14.34
The structure of lysozyme. Glu35 and
Asp52 are shown in white.
Substrate Analog Studies
• Natural substrates are not stable in the
active site for structural studies
• But analogs can be used - like (NAG)3
• Fitting a NAG of (NAG)6 into the D site
requires a distortion of the sugar
• This argues for stabilization of a transition
state via destabilization (distortion and
strain) of the substrate
Figure 14.35
(NAG)3, a substrate analog, forms stable
complexes with lysozyme .
Figure 14.36
The lysozyme -enzyme-substrate complex.
(Photo courtesy of John Rupley , University of
Arizona )
Figure 14.37
Enzyme-substrate interactions at
the six sugar residue-binding
subsites of the lysozyme active
site. (Illustration: Irving Geis. Rights
owned by Howard Hughes Medical
Institute. Not to be reproduced without
permission. )
The Lysozyme Mechanism
•
Studies with 18O-enriched water show that
the C1-O bond is cleaved on the substrate
between the D and E sites
• This incorporates 18O into C1 position of
the sugar at the D site, not into the
oxygen at C4 at the E site
1. Glu35 acts as a general acid
2. Distortion of the sugar ring at the D site
3. Asp52 stabilizes a carbocation
intermediate
Figure 14.38
The C1-O bond, not the O-C4 bond, is cleaved in the lysozyme
reaction. 18O from H218O is thus incorporated at the C1 position.
Figure 14.39
Two possible mechanisms for the
lysozyme reaction. In Path A, the
intermediate is a noncovalent
oxocarbenium ion (carbocation).
Path B depends upon a covalent
intermediate involving Asp52 and the
C-1 oxygen of the cleaved
glycosidic bond.
Figure 14.40
Mass spectra of lysozyme
complexes. (a) Wild-type hen
egg white lysozyme (HEWL).
(b) Mutant lysozyme with
Glu35 replaced with
glutamine [HEWL(E35Q)],
incubated with chitobiosyl
fluoride (NAG2F). (c) Wildtype HEWL, incubated with
2-acetamido-2-deoxy-b-Dglucopyranosyl-(14)-2deoxy-2-fluoro-b-Dglucopyroanosyl fluoride
(NAG2FGlcF). (d)
HEWL(E35Q), incubated
with NAG2FGlcF. Structures
of the species corresponding
to each peak observed in the
amss spectra are shown to
the right, with their expected
relatvie molecular mass.
(From Vocadlo,D.J.,et al., 2000.
Catalysis by hen egg-white
lysozyme proceeds via a
covalent intermediate. Nature
412:835-838.)
Figure 14.41
Stereo view of the ovalent NAG2FGlcF intermediate of the lysozyme reaction. (From
Vacadlo,D.J.,et al.,2000. Catalysis by hen egg-white lysozyme proceeds via a covalent
intermediate. Nature 412:835-838.)