Enzyme Mechanisms
Topics: TIM, Chymotrypsin, Rate Enhancement, Transition State
Complementarity
Important point of the day: The transition state, not the substrate itself, must be structurally compatible with the enzyme active site.
III. Triose Phosphate Isomerase , aka TIM - mechanism of action TIM is the enzyme that catalyzes the interconversion between DHAP (dihydroxyacetone phosphate) and GAP (glyceraldehyde-3-P) during glycolysis:
H
H
OH H O
H
H
O
O
P
O
-
DHAP
O-
O
TIM
H
O
H
H
O
P
O
-
GAP
O-
OH
The proposed mechanism of catalysis is as follows :
Glu165
C O
O
H
H
OH
His95
O H N
C
H
N
C
O H
O
H
O-H hyujgu
O H cis enediol
_
N
C
H
N
A. One piece of evidence that supports this mechanism is an experiment in which one of the substrate protons is replaced with tritium (T). When tritiated substrate undergoes the TIM-catalyzed reaction, it turns out that
1) the base (presumably in the enzyme active site) picks off the T and puts it back in a different place, and
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2) the reaction is stereospecific - the product is always pro-R, not pro-S.
O T
T H
OH
OH O
From this it was inferred that the reaction goes through an intermediate known as a cis-enediol, which allows a specific side of the compound to face the base:
H
OH
H
O
H
O
P
O
-
O
-
OH
B. Further evidence for the cis-enediol intermediate is provided by an experiment showing that a stable compound (below) with a structure like the proposed intermediate has a higher affinity for the enzyme than does the substrate. This implies that the compound resembles the transition state.
OH
N
O-
H
O
H
P
O
O
-
O
-
C. TIM efficiency displays a bell-shaped dependence on pH:
2
V
6.95
9.5
pH
As shown on the graph, the midpoints of the transitions occur at pH 6.95 and 9.5;
V max occurs in between at the peak. The transition points imply that 1) above pH
9.5, the enzyme loses activity by giving up a proton, or converting from an acid to a base, and 2) below pH 6.95, it loses activity by gaining a proton, or converting from a base to an acid. Therefore, TIM's catalytic mechanism appears to involve general acid-base catalysis .

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D. Yet another experiment further clarifies the mechanism. When the OH on
DHAP is replaced by the good leaving group Br, the Br leaves, and Glu165 subsequently forms a covalent bond in its place (see below). This pinpoints Glu165 as the base in TIM's active site responsible for the pH 6.95 end of the acid-base behavior .
Glu165
C= O
Br
H H
H
H
O
O
O
P
O
O
So what about the acid? The X-ray structure revealed that His 95 is also in the active site. However, His has a pKa of about 7, which is nowhere near the value of 9.5
expected from the V vs pH bell curve discussed above. A closer look at the enzymesubstrate complex shows that this discrepancy might be explained by the proximity of His to nearby atoms. It was shown that replacing Glu with its shorter counterpart Asp, thus changing the distance between the carbonyl and His95, decreases the pK of His95. Interactions with Cys at the end of one helix further support an effect of van der Waals interactions between His95 and nearby atoms on the pKa of His95. Thus, the orientation of residues in the enzyme active site is key for acid-base catalysis.
E. The crystal structure also illuminate other aspects of TIM's mechanism. For example, Lys 12, which probably actually contributes to the 9.5 pK observed in the pH rate profile, appears to be responsible for stabilizing the -ve charge of the transition state molecule.
A flexible loop "locks down" during catalysis, stabilizing the cis-enediol intermediate.
Note that:
H-bonds are strongest when the pKa's of the donor and acceptor groups are similar.
The negative charge on His95 is stabilized by its being at the amino-terminal end of a helix as well as by a positively charged Lys nearby.
At the end of the reaction, Glu165 goes back to being a base.

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(See Handout on Enzymes - Mechanisms of Action for figures and additional detail.)
II. Chymotrypsin is an enzyme that catalyzes hydrolysis of peptide bonds that have an aromatic (Phe, Tyr) or nonpolar R group on the amino side:
O R
O R
C N C C N C
H H
α
H H
α
H
2
O
Like TIM, chymotrypsin has an active site, in this case at the interface between its two domains. In contrast to TIM, chymotrypsin works by covalent catalysis, a mechanism characterized by the formation of a temporary covalent bond between the enzyme and the substrate.
Kinetics of covalent catalysis
Often when a covalent ES complex forms, one product (P1) is released rapidly while the other (S', precursor to P2) remains temporarily bound to the enzyme:
E + S ES k
2
ES'
+
P
1 k
3
E + P
2
This occurs in the case of chymotrypsin:
O
E-OH + R' C N R
H
ES
O
EO C R' covalent intermediate
Two types of evidence support this mechanism:
1) direct observation of the covalent intermediate
2) observation of 2 distinct phases of the reaction, an initial burst (dependent on k
>> k
3
2
) followed by a slower rate (dependent on k
3
). (For chymotrypsin, k
2
.) For this experiment, the compound paranitrophenylethylcarbonate is used as a substrate for chymotrypsin, and the appearance of the yellow product paranitrophenol is used to monitor the reaction. As [E] is increased, the initial burst of yellow is seen to increase in proportion to and eventually to approach [E]. See Handout p2, Fig 1, and V&V Fig 14-18 . For future reference, myosin hydrolysis works the same way.

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Mechanism - the catalytic triad - (See also V&V p. 390-1, Fig. 14-25 )
Chymotrypsin's active site contains three key residues:
Asp102 His57 Ser195
O
C
O
H N N H
O
C
H
The involvement of Ser195 was revealed by the finding that
diisopropylphosphofluoridate (DIPF) inactivates the enzyme by binding to Ser195.
However, the pKa of a normal Ser is ~17 and it should react very poorly with DIFP, suggesting that the OH on Ser has an unusually high reactivity. In a subsequent experiment, TPCK reacted with and thus revealed the presence of His57 in the active site, suggesting that His57 might deprotonate Ser-OH and thus make it more reactive. But Ser and His also have significantly different pKa's, suggesting that some third factor might be responsible for their interaction. Sure enough, X-ray crystallography revealed Asp102 at just the right distance to H-bond with the His57 imidazolium ion. So the story seemed complete: Asp102 is in a nonpolar environment and thus has a high enough pKa to pull an H off of His57, which in turn pulls an H off of Ser195, which then has a reactive O- which reacts with the substrate.
BUT molecular biology entered the picture and confused everyone. In a mutagenesis experiment, Asp102 was changed to an asparagine, which has no charge and was therefore expected to kill the enzyme based on the above model. As it turned out, the activity did decrease, but only from about 10 8 to 10 4 . Similarly, adding a methyl group to His and mutating Ser each decreased the activity but only partially. Thus, something else had to be going on.
A closer look at the structure of the active site indicates that changing the pKa of
Ser195, and hence the charge-dependent jobs of His57 and Asp102, is not as critical as originally thought. The precise orientation among residues in the active site and
their fit with the substrate intermediate is equally important. Key features include:
- a specificity pocket tailor-fit for aromatic R groups amino terminal to the
substrate's peptide bond (vs. trypsin, which cleaves after Lys or Arg)
- an oxyanion hole containing 2 amino acids to stabilize the O- formed when
Ser195 attacks the substrate, and shaped to fit the tetrahedral substrate
intermediate
- low barrier H-bonding - much stronger than regular H-bonds - because the
pKa's of the involved amino acids are all similar
These features of the active site structure lead to the fact that the interaction between the enzyme and the substrate in its transition state is stronger than the
interaction between the enzyme and the Michaelis-Menten (pre-reaction) substrate.
The fact that the transition state binds with greater stability to the enzyme than the

6 plain substrate contributes significantly to reaction rate enhancement - hence the unexplained leftover activity in the mutagenesis experiments. (see handout for whole mechanism)
III. General Mechanisms for Rate Enhancement.
A. Orientation An experiment to support the importance of orientation for catalytic efficiency (that is, for rate enhancement), is shown on p5 of the handout,
Figure B. Here positioning the reactive groups more closely can speed up the rate by 5 x 10 4 times. This shows that orientation alone is also not sufficient to achieve the observed 10 8 rate enhancement seen for many enzymes. Where does the rest of the rate enhancement come from?
B. Transition State Stabilization On p4 of the handout, paragraph B, it is shown that one can change the substrate for the enzyme pepsin, which prefers to cleave hydrophobic peptides. As the substrates become more hydrophobic the K
M
for these substrates actually only varies a little, but the value of kcat decreases by 100fold - i.e. K
M
stays the same, but Vmax changes. If K
M
is a reflection of the substrate's affinity for an enzyme, then why is K
M
not the value that changes?
These results suggest that, in fact, part of the excess binding energy available from the more hydrophobic substrates is actually being used to enhance the rate of the reaction.
The binding energy between the substrate and the enzyme is partially used to constrain the substrate into the conformation of the transition state.
Does this work to enhance the rate? (see handout pg. 6, paragraph 2 to end, and below)
For an enzyme catalyzing a reaction from S to P, a thermodynamic box can be written that describes the reaction leading to P both with and without binding to the enzyme. The top reaction shows the reaction reaching the transition state when S is not bound to E and the lower reaction is for when S is bound to E. The equilibrium constants for reaching the transition state, S ‡ , and the dissociation constants for S or
S ‡ binding to the enzyme are then related as given below. (See V&V p. 380 for detail).
K
N
E + S
K
S
E + S
K
T
E + P
ES
K
E
ES E + P
K
E
K
N
=
K
K
T
S
This implies that the enzyme would bind preferentially to the transition state, since the catalyzed reaction occurs faster than the uncatalyzed reaction. That is, the dissociation constant K
T
would be smaller than K
S
, because the equilibrium constant for reaching the transition state on the enzyme, K
E
‡ , is larger than the equilibrium constant for reaching the transition state without being bound to the enzyme, K
N
‡ .

Experiment: make transition state analog and see how tightly it binds to E. It turns out to bind much more tightly to E; affinity is 10 2 - 10 4 times that of substrate.
So, in summary, enzymes are designed with two features:
- arranged so catalytic sites are oriented correctly
- have an active site that complements the transition state and thus lowers the activation energy for getting to the transition state (by ∆ activation energy in figure)
∆ activation E
G
S
P
Validation:
1) The more an analogue is like the transition state, the higher is kcat/K
M.
2) Antibody catalysts - antibodies generated against the transition state contort substrate into the transition state conformation, resulting in hydrolysis.
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