The hydrolysis of carbobenzoxy-L-tyrosine p-nitrophenyl ester by various enzymes.

cY-Chymotrypsin rapidly catalyzes the release of p-nitrophenol from carbobenzoxy-n-tyrosine p-nitrophenyl ester and the reaction has been made the basis of a spectrophotometric assay requiring only millimicrogram quantities of the crystalline enzyme (1). Other nitrophenyl esters, e.g. of acetic acid (2), hydrocinnamic acid (3), and carbobenzoxyglycine (4) are also hydrolyzed by chymotrypsin despite the absence of structural parameters previously considered requisite in substrates sensitive to thii enzyme (5). As a generalization, an increase in the rate of the deacylation reaction is observed as the acyl contributor to the sensitive bond approaches the structure of an aromatic amino acid residue (3, 6). Trypsin has also been reported to hydrolyze p-nitrophenyl acetate (6), a compound of great structural variance to other trypsin-sensitive synthetic substrates (5). The hydrolysis of this substrate by both chymotrypsin and trypsin represents another instance of the cross-reactivity of these two enzymes to the same or closely related substrates (T-11). Other enzymes such as erythrocyte cholinesterase (12), eel cholinesterase (13), and the A-, B-, and C-esterases (14-16) are also capable of hydrolyzing p-nitrophenyl acetate and related esters, e.g. phenyl acetate. Since we are interested in the possible use of acylated aromatic amino acid esters containing an aromatic alcohol as contributor to the sensitive bond for the assay of proteolytic enzyme activity in very small tissue samples, it was considered appropriate to determine if enzymes other than chymotrypsin could hydrolyze carbobenzoxy-n-tyrosine p-nitrophenyl ester. Preliminary reports of some of this material have appeared (17, 18).