The Chymotrypsin MechanismInvolves Acylation and Deacylationof a Ser Residue
Bovine pancreatic chymotrypsin is a protease, anenzyme that catalyzes the hydrolytic
cleavage of peptide bonds.This protease is specific for peptide bonds adjacent to aromatic
amino acid residues (Trp, Phe, Tyr).
The reaction catalyzed by this enzyme illustrates the principle of transitionstate stabilization
and also provides a classic example of general acid-base catalysis and covalent catalysis.
Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109
. . The reaction thus has two distinct phases. In the acylation phase, the peptide bond is
cleaved and an ester linkage is formed between the peptide carbonyl carbon and
the enzyme. In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated
enzyme is regenerated.
The nucleophile in the acylation phase is the oxygen of Ser195. (Proteases with a Ser
residue that plays this role in reaction mechanisms are called serine proteases.) However,
in chymotrypsin, Ser195 is linked to His57 and Asp102 in a hydrogen-bonding network
referred to as the catalytic triad. When a peptide substrate binds to chymotrypsin, a subtle
change in conformation compresses the hydrogen bond between His57 and Asp102
, resulting in a stronger interaction, called a lowbarrier hydrogen bond. This enhanced
interaction increases the pKa of His57 from ∼7 to >12, allowing the His residue to act as an
enhanced general base that can remove the proton from the Ser195 hydroxyl group.
Deprotonation preventsdevelopment of a highly unstable positive charge on the Ser195
hydroxyl and makes the Ser side chain a stronger nucleophile. At later reaction stages,
His57 also acts as a proton donor, protonating the amino group in the displaced portion of
the substrate ). As the Ser195 oxygen attacks the carbonyl group of the substrate, a very
short-lived tetrahedral intermediate is formed in which the carbonyl oxygen acquires a
negative charge. This charge, forming within a pocket on the enzyme called theboxyanion
hole, is stabilized by hydrogen bonds contributed by the amide groups of two peptide bonds
in the chymotrypsin backbone. The intermediate collapses in step , breaking the peptide
bond. The amino group of the first product is protonated by His57, now acting as a general
acid catalyst. Water is the second substrate, entering the active site in step . As water
attacks the carbon in the ester linkage in step , and the resulting intermediate collapses to
break the ester linkage and generate the second product in step , His57 again acts — first as
a general base to deprotonate the water, and then as a general acid to protonate the Ser
oxygen as it leaves. Dissociation of the second product ) completes the reaction cycle.
