Inactivation of alpha-chymotrypsin by a bifunctional reagent, 3,4-dihydro-3,4-dibromo-6-bromomethylcoumarin.

Abstract

α‐Chymotrypsin is rapidly and irreversibly inactivated by 3,4‐dihydro‐3,4‐dibromo‐6‐bromo‐methylcoumarin (VII) at neutral pH. The inactivation is pH dependent between pH 5 and 7 and is delayed when the active site of the enzyme is protected by acetylation of the active serine‐195 or by binding of a competitive inhibitor.This dihydrocoumarin VII and its 6‐methyl analogue are substrates for α‐chymotrypsin, but when the ester bond in VII is broken during formation of the acyl‐enzyme, a reactive p‐hydroxybenzylbromide group is generated in situ within the active site. This newly formed group then alkylates one histidine residue (probably histidine‐57), as shown by amino acid analysis of the modified enzyme on acid hydrolysis. A model non‐enzymic reaction shows that the dihydrocoumarin VII is able to alkylate imidazole in aqueous solution at pH 7, yielding the N‐substituted imidazole, 3‐bromo‐6‐(imidazol‐1‐yl‐methyl)coumarin (XI).The modified enzyme has practically no activity against a specific substrate and seems no longer to have its intact active site. However its binding site is at least partly free for it is still able to bind proflavin.α‐Chymotrypsin is also inactivated by 6‐bromomethylcoumarin, the ester bond of which is stable, but the mode of inactivation and the properties of the modified enzyme are different from those found in the study with the dihydrocoumarin VII.