Alpha-chymotrypsin (Fig. 2) catalyzes the facile hydrolysis of peptide bonds, in particular those adjacent to the carboxyl group of aromatic amino acids (tryptophan, tyrosine, phenylalanine) as well as a variety of esters derived from similar N-acylated amino acids. The enzyme has been the subject of intensive mechanistic study, most of which occurred well before a crystal structure was available.
A key insight was provided by studying the enzymecatalyzed hydrolysis of p-nitrophenyl acetate. Transient kinetic studies revealed burst kinetics (Fig. 3) with an initial rapid liberation of p-nitrophenolate followed by a slower steady-state rate. The biphasic time course is consistent with the existence of two intermediates (ES and acyl-E), with the second accumulating owing to its slower breakdown to product. The intermediate is a covalent enzyme species acylated at serine-195 (see Fig. 2), a fact initially revealed by chemically esterifying this enzyme residue specifically and irreversibly with diisopropylphosphorofluoridate. No burst kinetics is seen with amide substrates because the acylation step limits turnover. The same intermediate, however, is formed as shown by partitioning experiments in which an exogenous nucleophile such as hydroxylamine is added to compete with water in the deacylation step. The result revealed equivalent levels of hydroxamate and acid products formed from either amide or ester substrates derived from a common amino acid, which implicated the presence of the intermediate in both enzyme-catalyzed processes.
The pH dependence of the steady-state kinetic parameters is shown in Fig. 4 and implicates the ionization of two groups in the free enzyme and one in the ES complex. These data combined again with chemical modification studies (now superseded by site-specific mutagenesis) implicated histidine-57 (pKa ~ 7) and the N-terminal amino acid isoleucine (pKa ~ 8.5). The latter forms a salt bridge with aspartate-194 that helps maintain the active structure of the enzyme; the former is involved in general acid–base chemistry at the active site.
These data, along with further information derived from the reaction of specific substrates with the enzyme by using stopped-flow methods, led to the elucidation of a kinetic sequence that consistently implicated the acylation and deacylation of Ser195 assisted by His57 and Asp102. The crystal structure of chymotrypsin (Fig. 2) reveals that these three residues form a catalytic triad, a feature repeated for many hydrolytic enzymes. This triad operates within a well-defined binding site that is lined with nonpolar amino acids capable of van der Waals interactions with polypeptide substrates containing aromatic side chains. A plausible mechanism is outlined in Fig. 5 in terms of the chemistry occurring during the individual kinetic steps.
The key features of this mechanism require the participation of the serine hydroxyl as a nucleophile whose attack on the carbonyl of the substrate is facilitated through proton abstraction by the imidazole nitrogen of His57 and its redonation to the amine-leaving group. Deacylation of the enzyme follows general base catalysis of water attack again by His57 and the return of the enzyme to its resting state. Catalysis of the chemical process through the participation of the side chains of an enzyme in proton, hydride, and electron transfer is a hallmark of enzyme catalysis and can occur efficiently in the confines of the active site owing to the optimal alignment and juxtapositioning of the substrate for chemical reaction.
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