The Kinetics of α-Chymotrypsin Reactions in the Presence of Added Nucleophiles

Abstract

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.

Serine carboxypeptidases. A review

“…an amine compound, a partitioning of the acyl-enzyme may take place to yield a hydrolysis product and an aminolysis product. BENDER et al (13) and FASTREZ and FERSHT (53) found that when methanol, ethanol, glycine amide or alanine amide were added as nucleophiles to chymotrypsin it reacted with ester substrates according to the simple reaction model shown in Scheme 6, in which it is assumed that neither water nor the added nucleophile bind to the enzyme prior to the reaction with the acyl-enzyme intermediate. Hence, the ratio of the two reaction products being formed,…”

Malt carboxypeptidase catalyzed aminolysis reactions

“…amino compounds, the acylenzyme intermediate will react both with water to give a hydrolysis product and with the amine to give an aminolysis product. BENDER et al ( 1 ) and FASTREZ and FERSHT (9) found that when methanol, ethanol, glycine amide or alanine amide were added as nucleophiles to chymotrypsin it reacted with ester substrates according to the simple reaction model shown in Scheme ~[w] Scheme 1. Competition between water (W) and other nucleophile (N) in the serine protease catalyzed hydrolysis of ester substrates as proposed by BENDER et al (1).…”

N‐α‐benzoyl arginine ethyl ester hydrolysis by procine plasmin. A kinetic study of diethyleneglycol influence on this hydrolysis

“…Since DEG influence could be suspected of being nucleophilic, Bender's simplest equation was examined [19]:…”