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T h e effect of added nucleophiles such as methanol, ethanol, and hydroxylamine on the kinetics of a-chymotrypsincatalyzed reactions is interpreted in terms of the competitive partitioning of the acyl-enzyme intermediate by water and the added nucleophile according to eq. 2. In agreement with this hypothesis, the rate of deacylation of trans-cinnamoyl-a-chymotrypsiri in methanol-water solutions is dependent on the methanol concentration, and the products of the reaction are both methyl cinnamate and cinnamate ion. Data a t high methanol concentrations do not indicate a saturation phenomenon, thus giving no evidence for a binding of methanol (or water) to the enzyme. The a-chymotrypsin-catalyzed hydrolysis and alcoholysis of both specific and nonspecific substrates of a-chymotrypsin conform to the kinetics predicted by eq. 2. The kinetics of hydrolysis and methanolysis of N-acetyl-L-tyrosineamide and of N-acetyl-L-phenylalanineamide must be explained on the basis of eq. 2. A complete set of,rate constants can be calculated from the alcoholysis experiments for the specific substrates, N-acetyl-L-tryptophan ethyl and methyl esters and N-acetyl-L-phenylalanine methyl ester. The a-chymotrypsin-catalyzed hydrolysis and hydroxylaminolysis of N-acetyl-L-tyrosine ethyl ester and N-acetyl-L-tyrosine hydroxamic acid may be interpreted in terms of an expansion of eq. 2, eq. 16, which takes into account both the enzymatic and nonenzymatic fates of the labile initial product 0-( N-acetyl-Ltyrosy1)hydroxylamine.

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The pH dependencies of the kinetic constants for the dephosphorylated pepsin catalyzed hydrolysis of N-acetyl-L-phenylalanyl-L-tyrosine methyl ester were determined in the pH range 1.12-5.39 at 25°. The dephosphorylated pepsin was prepared by enzymatically dephosphorylating pepsinogen with potato phosphatase at pH 5.60 and then activating this pepsinogen using the procedure of Rajagopalan, Moore, and Stein. The pH vs. k,;it curve is bell-shaped and depends on a pair of catalytic groups with p7sfa's of 2.1 and 4.6. The catalytic behavior of this dephosphorylated pepsin is compared to that of "regular" pepsin. A discussion of the role of and type of bonding of this phosphate group in pepsin concludes the paper.