2. Two Substrate Reactions
Many enzyme reactions involve two or more substrates. Though the Michaelis-Menten equation was derived from a single substrate to product reaction, it still can be used successfully for more complex reactions (by using kcat).
Random
Ordered
Ping-pong

3. Two Substrate Reactions
In random order reactions, the two substrates do not bind to the enzyme in any given order; it does not matter which binds first or second.
In ordered reactions, the substrates bind in a defined sequence, S1 first and S2 second.
These two reactions share a common feature termed a ternary complex, formed between E, ES1, ES2 and ES1S2. In this situation, no product is formed before both substrates bind to form ES1S2.

4. Two Substrate Reactions (cont)
Another possibility is that no ternary complex is formed and the first substrate S1 is converted to product P1 before S2 binds. These types of reactions are termed ping-pong or double displacement reactions.

5. Principles illustrated: Transition-state stabilization;
The catalytic mechanism of chymotrypsin: a member of the serine protease family; catalyzes the hydrolytic cleavage of peptide bonds adjacent to aromatic amino acid residues (with a rate enhancement of at least 109).
Principles illustrated:
Transition-state stabilization;
General acid-base catalysis;
Covalent catalysis.

6. Chymotrypsin (and other proteins) are activated via proteolytic cleavage of precursor proteins (zymogens or preproteins). Many proteases activated this way can be inactivated by inhibitor proteins tightly-bound in the active sites.

11. Active chymotrypsin and trypsin are produced from inactive zymogens via proteolytic cleavage, with
conformational changes exposing the active sites.

12. The catalytically important groups of chymotrypsin were identified by chemical labeling studies
Organic fluorophosphates such as diisopropylphosphofluoridate (DIPF) irreversibly inactivate chymotrypsin (and other serine proteases) and reacts only with Ser195 (out of the 25 Ser residues).

13. A second catalytically important residue, His57, was discovered by affinity labeling with tosyl-L-phenylalanine chloromethylketone (TPCK)
• TPCK alkylates His 57
• Inactivation can be inhibited by b-phenylpropionate (competitive inhibitor)
• TPCK modification does not occur when chymotrypsin is denatured in urea.

14. Rapid initial burst kinetics indicates an acyl-enzyme intermediate
The kinetics of chymotrypsin is worked out by using artificial substrates (esters), yielding spectroscopic signals upon cleavage to allow monitoring the rate of reactions.
Colorless substrate
Yellow product
Fast
This reaction is far slower
than the hydrolysis of peptides!
Slow
Km = 20 mM
Kcat = 77 s-1

15. The catalysis of chymotrypsin is biphasic as revealed
by pre-steady state kinetics
Slow phase (enzymes will be
able to act again only after a slow
deacylation step)
“burst” (fast) phase (rapid acylation of all
Enzymes leading to release of p-nitrophenol)
Milliseconds after mixing

16. Determination of the crystal structure of chymotrypsin (1967) revealed a catalytic triad: Ser195, His57, Asp102.

17. Chymotrypsin: three polypeptide chains linked by multiple disulfide
bonds; a catalytic triad.
Active site
His57
Ser195
Asp102
Cleft for binding
extended substrates
Trypsin, sharing a 40% identity with
chymotrypsin, has a very similar structure.

18. A catalytic triad has been found in all serine proteases: the Ser is thus converted into a potent nucleophile

19. The Peptide Bond has partial (40%) double bond character as a result of resonance of electrons between the O and N
The hydrolysis of
a peptide bond
at neutral pH
without catalysis
will take ~
years!
This is the peptide group which remains planar

20. Chymotrypsin (and other serine proteases) acts via a mixture of covalent and general acid-base catalysis to cleave (not a direct attack of water on the peptide bond!)

21. Formation of the ES complex E S Formation of ES1 ES1
Asp102 functions only to orient His57.
Formation of the
ES complex E S
Formation of ES1
ES1
The peptide bond to be cleaved is positioned by the binding of the side chain of an adjacent hydrophobic residue in a special hydrophobic pocket.

22. His57 acts as a general base in
deprotonating Ser195, the alkoxide
ion then acts as a nucleophile,
attacking the carbonyl carbon.
ES1
Ser195 forms a covalent bond
with the peptide (acylation) to
be cleaved. a trigonal C is
turned into a tetrahedral C.
The tetrahedral oxyanion
intermediate is stabilized by
the NHs of Gly193 and Ser195
Pre-acylation
Preferential binding of the transition state: oxyanion hole stabilization of the negatively charged tetrahedral intermediate of the transition state.
oxyanion hole

23. ES1 Acylation Acyl-E Releasing of P1 His57 acts as a general acid
in cleaving the peptide bond.
ES1
Acylation
Releasing of P1
The amine product is
then released from the
active site with the
formation of an acyl-enzyme
covalent intermediate.
Acyl-E

24. E’S2 Acyl-E Entering of S2 Water (the second substrate)
then enters the active site.

25. E’S2 Pre-deacylation His57 acts as a general base
again, allowing water to attack
the acyl-enzyme intermediate,
forming another tetrahedral
oxyanion intermediate, again
stabilized by the NHs of Gly193
and Ser195 (similar to step 2)
Pre-deacylation
E’S2

26. EP2 His57 acts as a general acid again in breaking the covalent
bond between the enzyme
and substrate (deacylation)
(similar to Step 3).
Deacylation

27. E EP2 Recovered enzyme Release of P2 The second product
(an acid) is released
from the active site,
with the enzyme recovered
to its original state.
EP2

28. E EP2 ES E’S2 Acyl-E Deacylation phase Acylation phase
2nd product
1st substrate E
EP2 ES
The proposed complete
catalytic cycle of
chymotrypsin
(rate enhancement: 109)
A Ping-Pong Mechanism
1st product
E’S2
Acyl-E
Deacylation
phase
Acylation
phase
2nd substrate