The inactivation of papain and glyceraldehyde-3-phosphate dehydrogenase by phenyldiimide

Abstract

Phenylhydrazine does not inactivate papain or glyceraldehyde-3-phosphate dehydrogenase under anaerobic conditions. The inactivation of papain and glyceralde-hyde-3-phosphate dehydrogenase under aerobic conditions is ascribed to the oxidation of phenylhydrazine by O 2 which generates phenyldiimide and H 2O 2, both of which react with the essential sulfhydryl groups and inactivate the enzymes. Phenyldiimide generated from methyl phenylazoformate inactivates both of the sulfhydryl enzymes under anaerobic conditions. The inactivation of papain and GPD with aerobic, aqueous solutions of [ 14C]phenylhydrazine introduces a small amount of radioactivity into the enzymes which is discharged by dithiothreitol. The amount of radioactivity bound to papain is increased when cyanide is present in the inactivation mixture.When the β-[ 14C]thiocyanoalanine derivative of papain is treated with phenylhydrazine the radioactivity is discharged from the enzyme. Cyanide evidently reacts with the sulfenic acid derivative of papain to form a thiocyanate derivative. Phenylhydrazine presumably displaces cyanide from the thiocyanate derivative to form a sulfenyl hydrazide derivative to account for the increased incorporation of [ 14C]phenylhydrazine when papain is inactivated with aerobic solutions of [ 14C]-phenylhydrazine in the presence of cyanide. When the sulfhydryl group of papain is oxidized to a sulfenic acid with H 2O 2 and then treated with [ 14C]phenylhydrazine, 14C is not incorporated into the enzyme. These experiments suggest that the H 2O 2 in the aerobic solutions of phenylhydrazine oxidizes the sulfhydryl group at the active site of papain to a sulfenic acid. The [ 14C]phenyldiimide in these solutions reacts to some extent with the active sulfhydryl group to form a sulfenyl hydrazide derivative.