Evidence for the Formation of Hippuryl Chymotrypsin during the Hydrolysis of Hippuric Acid Esters

The formation of an acyl enzyme derivative as an intermediate in the enzymic hydrolysis of esters was first suggested for the enzyme acetylcholinesterase (1). This theory offers a ready explanation for a number of diverse phenomena including transacylation reactions (l), oxygen exchange between acids and water (2-4), the existence of a noncompetitive component in the reversible inhibition produced by substituted ammonium ions (5, 6), and substrate inhibition (6, 7). It explains the formation of phosphoryl @-lo), carbamyl (ll), and sulfonyl enzyme derivatives (12, 13). It suggested the means of reactivating the alkyl phosphate-inhibited enzyme (10). In the case of chymotrypsin, several acyl enzymes have been demonstrated after reaction with p-nitrophenyl esters or other anhydrides and some have been isolated (14-18). The important studies that report on these enzymes do not quite settle the question because p-nitrophenyl esters are active acylating agents, and it has been found that the mechanism of the general basecatalyzed hydrolysis of anhydrides differs from that of ordinary esters in that acyl transfer is involved with the anhydrides (19). Although it is hydrolyzed by chymotrypsin, p-nitrophenyl acetate, for example, is about lo4 times less rapidly acted upon than the esters of suitable amino acid derivatives (20, 21). There might be, therefore, some question as to whether the compound is acting as an acylating agent or as a substrate. There might be some question whether the same site is involved and if it is, some doubt whether normal substrates react in quite the same manner. Some recent work has shown that. esters of cinnamic acid, which are substrates for chymotrypsin, though very poor ones, react with the enzyme to produce spectral changes that, with the arguments and other evidence presented, strongly indicate the formation of a cinnamoyl enzyme derivative (22). The body of evidence for the formation of an acyl enzyme intermediate in chymotrypsin-catalyzed reactions is thoroughly reviewed by Bender (23). The earlier evidence from acetylcholinesterase-catalyzed reactions is reviewed in The Enzymes