The activation of papain and the inhibition of the active enzyme by carbonyl reagents.

The activation of papain with four different activators is not accompanied by the binding of any of them to the protein. These experiments, taken together with previously reported results, show that the inactive form of papain prepared by the method of Kimmel and Smith (J. Biol. Chem., 207, 5’75 (1954)) is a mixed disulfide formed between the active site sulfhydryl group of the protein and free cysteine. The known inhibition of the activated enzyme by reagents having affinity for carbonyl groups has been investigated in order to determine whether an aldehyde residue which is intimately connected with the activation process is present on the enzyme, or if the observed inactivation of the protein by this class of reagents can be accounted for in some other manner. The following relevant observations were made. (a) Phenylhydrazine inactivates and binds to cyanide-activated papain but neither inactivates nor binds to cysteineor borohydride-activated papain in the presence of excess activator. (b) The cysteine-activated enzyme is also inhibited by phenylhydrazine when the cysteine to papain ratio is low. (c) This inhibition in the presence of cyanide or low concentrations of cysteine is readily reversible with excess cysteine which also releases the bound phenylhydrazine from the protein. (d) Treatment of the enzyme with either phenylhydrazine or hydrogen peroxide in the presence of WNresults in the binding of one cyanide group per active site on the enzyme. Carboxamidomethylation of the sulfhydry1 group at the active site prevents this reaction. (e) Semicarbazide and hydroxylamine, other reagents having high affinity for the carbonyl group, are much less effective inhibitors of papain. (f) Reactivation of cyanide-activated, peroxide-inactivated papain by cysteine yields Z-iminothiazolidine-4-carboxylic acid. Model studies show that phenylhydrazine, but not semicarbazide or hydroxylamine, is capable of oxidizing cysteine to cystine during 1 hour of incubation with 30 mM reagent. Phenylhydrazine does not bind to cyanate-inactivated papain. Activator-free papain is irreversibly inhibited by phenylhydrazine, without concomitant binding of the reagent. These and other experiments show that the inhibition of cyanide-activated papain by carbonyl reagents is not due to the affinity of these compounds for a carbonyl group on the enzyme, but rather to an oxidative coupling of cyanide to the enzyme resulting in the