THE ACTIVE SITE IN α-CHYMOTRYPSIN: METHYL 3,4-DIHYDROISOCOUMARIN-3-CARBOXYLATE I

Publisher Website
Google Scholar
Abstract
DL-Methyl 3,4-dihydroisocoumarin-3-carboxylate (I) has been prepared. It is hydrolyzed rapidly with preferred D-specificity by [alpha]-chymotrypsin; kcat/Kmapp is estimated to be 4 x 104 M -1 sec-1 for the D enantiomorph; the Lenantiomorph is less reactive. The reactivity of D-I, despite the absence of an amide group, is essentially the same as that of its N-analogue, D-l-keto-3-carbomethoxytetrahydroiso-quinoline, (D-IV), and similar to that of L-ethyl-N-acetyl-[beta] -phenyl-alaninate (II). The NH group in D-IV has no specific function. The aroyloxy group of I and the aroylamido group of IV bind at the ar [aryl group] site of the enzyme, and D-I and D-IV do not utilize the am [acyl amido group] site, leading to D specificity. The hetero-cyclic rings of I and IV lead to high reactivity by restricting rotations at C[alpha]-C[beta] and C[beta] -C-phenyl, and they place the carbalkoxyl group, in its equatorial conformation, at the n site when the aryl group binds at ar. In the natural substrate II. the [alpha]-acetamido group binds at am, leading, with the binding of the [beta] -phenyl at ar, to equivalent restriction of the substrate. This is the major activating function of the [alpha]-acylamido group. The similar high reactivities of D-I, D-IV, and L-II indicate that distortion of substrate is unimportant in hydrolysis by [alpha]-chymotrypsin, and that reorganization of the enzyme, if it occurs, results from binding at ar.