Inactivation of α-chymotrypsin by a bifunctional reagent, 2-bromomethyl-3,1-benzoxazin-4-one

Abstract

α-Chymotrypsin is selectively and irreversibly inactivated by 2-bromomethyl-3, i-benzoxazin-4-one (Compd Ib) at neutral pH. Reaction of the enzyme with this activated ester leads rapidly to a relatively stable acyl-enzyme in which intramolecular alkylation of a single methionine residue (likely methionine-192) followed by hydrolysis of the acyl-enzyme bond occurs. Kinetic evidence for such a process is found in the measurements of proflavin displacement accompanying the reaction. The irreversibly modified enzyme still possesses its intact active site but its activity towards specific substrates is altered. Alkylation of the enzyme increases mainly K m but does not change appreciably k cat. The inhibition constants of specific inhibitors such as proflavin or indole are increased several times. From crystallographic data it is known that methionine-192 forms the lid of the specificity cavity in the enzyme. Therefore a bulky substituent such as the 2-acetamido benzoic acid on the sulfur atom of this methionine might sterically hinder the substrate binding by blocking the approach of the binding site.