Specificities of α-Chymotrypsin and Subtilisin Carlsberg: THE α-ACYLAMIDO EFFECT IN β-PHENYLPROPIONATES AND THEIR RIGID ANALOGS

Abstract

Abstract A study of the comparative specificities of α-chymotrypsin and subtilisin Carlsberg has been focused mainly on the extent to which α-acetamido groups of some specific and nonspecific activated ester substrates affect the reactivity of each enzyme. Comparisons are based upon kc and kc:Km corrected for substrate intrinsic reactivities ((kc)n and (kc:Km)n). Among p-nitrophenyl β-phenylpropionates (α substituents: NHCOCH3, H, O2CCH3), (kc:Km)n of the specific substrate N-acetyl-(S)-phenylalanine p-nitrophenyl ester ((S)-I) is 16 times higher than for p-nitrophenyl β-phenylpropionate (II); in (kc)n (S)-I is hydrolyzed 110 times faster than II. With subtilisin Carlsberg no detectable enhancement attributable to the α-acetamido group of the substrate was found in (kc:Km)n of (S)-I. With each enzyme the use of activated esters is largely responsible for the lack of a substantial α-acetamido effect upon (kc:Km)n since in (kc:Km)n of the methyl esters corresponding to (S)-I and II, the enhancement is about two orders of magnitude. The investigation was extended to include the p-nitrophenyl esters of 1,2,3,4-tetrahydro-2-naphthoic and indan-2-carboxylic acids (α substituents:NHCOCH3, H, O2CCH3). The α-acetamidosubstituted member of each of the two series of semirigid substrates is constrained in a geometry similar to that postulated as the conformation of (S)-I preferred during hydrolysis by chymotrypsin and subtilisins. With both enzymes a substrate α-acetamido group-enzyme interaction makes the S isomer of p-nitrophenyl 2-acetamido-1,2,3,4-tetrahydronaphthalene-2-carboxylate ((S)-IV) two orders of magnitude more reactive than its unsubstituted derivative. A similar interaction does not enhance the reactivity of p-nitrophenyl 2-acetamidoindan-2-carboxylate (VII). The reactivities of α-chymotrypsin and subtilisin Carlsberg toward the α-acetoxy derivatives of each substrate type are lower than those exhibited toward their α-acetamido and unsubstituted derivatives.