Specificities of &hymotrypsin and Subtilisin Carl&erg THE a-ACYLAMIDO EFFECT IN &PHENYLPROPIONATES AND THEIR RIGID ANALOGS*

A study of the comparative specificities of cr-chymotrypsin and subtilisin Carlsberg has been focused mainly on the extent to which a-acetamido groups of some specific and nonspecific activated ester substrates affect the reactivity of each enzyme. Comparisons are based upon k, and k,: K,,, corrected for substrate intrinsic reactivities ((k,), and (k,: K&J. Among p-nitrophenyl P-phenylpropionates (ar substituents: NHCOCH,, H, OsCCH8), (k,:K,), of the specific substrate iV-acetyl-(S)-phenylalanine p-nitrophenyl ester ((S)-I) is 16 times higher than for p-nitrophenyl S-phenylpropionate (II); in (k,), (S)-I is hydrolyzed 110 times faster than II. With subtilisin Carlsberg no detectable enhancement attributable to the a-acetamido group of the substrate was found in (k,: K,), of (S)-I. With each enzyme the use of activated esters is largely responsible for the lack of a substantial cu-acetamido effect upon (k,: K,), since in (k,: K,), 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 (a! substituents:NHCOCHB, H, 02CCH3). The a-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 a-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 cY-chymotrypsin and subtilisin Carlsberg toward the cr-acetoxy derivatives of each substrate type are lower than those exhibited toward their ac-acetamido and unsubstituted derivatives.