Inactivation of chymotrypsin by diphenyldiazomethane.

a-Chymotrypsin is a protein composed of three peptide chains linked by disulfide bridges, with amino-terminal half-cystine, isoleucine, and alanine residues.1 The latter two end groups appear upon the enzymic conversion of chymotrypsinogen A (which only contains the amino-terminal half-cystine residue) to a-chymotrypsin. The specificity of a-chymotrypsin as an enzyme (and of other chymotrypsins derived from chymotrypsinogens A and B) is preferentially directed to the cleavage of substrates in which the CO group of sensitive amide or ester bonds is donated by an aromatic L-amino acid residue (tryptophan, phenylalanine, tyrosine); lower catalytic efficiency is exhibited toward bonds in which the CO group is donated by other L-amino acids bearing apolar side chains (methionine, leucine, etc.).2 The available evidence strongly supports the view that the mechanism of chymotrypsin action on suitable amide and ester substrates [e.g., acetyl-L-tyrosinamide, acetyl-L-tyrosine ethyl ester (ATEE) ]3 involves the formation of an acyl enzyme4 (e.g., acetyltyrosyl-chymotrypsin), and that the site of acylation is the f3-hydroxyl group of a serine residue (no. 195 in the amino acid sequence of chymotrypsinogen A).5 Studies on the pH dependence of the catalytic action of chymotrypsin,6 and on the action of inhibitors such as tosyl-L-phenylalanyl chloromethane7 indicate the participation of a histidine residue (histidine 57), with the possible involvement of the other histidine residue (histidine 40), in the acylation and deacylation of the reactive serine hydroxyl group. These two histidine residues are held near each other by a disulfide bridge linking half-cystine residues 42 and 58.8 The ready acylation of the reactive serine by p-nitrophenyl acetate9 or by cinnamoyl imidazole,'0 and the relative stability of the acyl enzymes so formed, have permitted studies leading to notable progress in the description of the mechanism of chymotryptic catalysis.11 These studies have also brought out the striking difference in the rates of deacylation of acyl enzymes derived from reaction with a specific substrate (e.g., ATEE) and from reaction with an acylating agent such as p-nitrophenyl acetate; the replacement of the acetyl group of acetyl-chymotrypsin by an acetyl-L-tyrosyl group causes an enhancement of about 12,000-fold in the rate of deacylation. This difference can be interpreted most readily as evidence of the effect of specific binding of the acetyltyrosyl group at a "binding site" located in the vicinity of the "catalytic site" of the enzyme. Although considerable information is available about the amino acid residues that participate in the action at the catalytic site (serine 195, histidine 57, possibly histidine 40), there is less knowledge about the nature of the amino acid side chains that are involved in the formation and maintenance of the specific binding site of chymotrypsin. Studies on the effect of oxidationl2 or alkylation'3 of a-chymotrypsin have indicated that chemical modification of one or both of the two methionine residues (180 and 192) alters the binding site to a greater extent than the catalytic site. Furthermore, evidence has been presented'4 in favor of the view that the