Specific and irreversible inactivation of pepsin by substrate-like epoxides.

Several substrate-like epoxides were found to act as specific and irreversible inactivators of pepsin. Of these, 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) was the most potent. When EPNP reacted with pepsin, all enzyme activity was lost. Two molecules of EPNP were found to be covalently bound to each molecule of completely inactivated enzyme. In this reaction, two carboxyl groups of the enzyme are each apparently esterified by one hydroxyl group of glyco1 formed from EPNP. The esterification of carboxylates by epoxides is a well established reaction. The modified enzyme, EPNP-pepsin, released the bound EPNP groups in alkaline solution. On thin layer chromatography, the released modifying group was identified as the glycol of EPNP. These observations support the view that esterification was the inactivation reaction. Prior reaction of pepsin with diazoacetyl-DL-norleucine methyl ester or p-bromophenacyl bromide was still followed by the reaction of 2 EPNP residues per pepsin molecule. The EPNP-reactive carboxyl group must, therefore, differ from those carboxyl groups reacted with diazoacetyl-DLnorleucine methyl ester and p-bromophenacyl bromide. EPNP failed to inactivate pepsinogen. Moreover, in the presence of synthetic substrates or their hydrolytic products, the inactivation of pepsin by EPNP was significantly slower. Results on the EPNP incorporation in the presence and absence of substrates indicated that only one of the two modified carboxyl groups was protected by the presence of the dipeptide substrate. This “active center carboxyl group” may participate in the catalysis of the enzyme. The second EPNP-reactive carboxyl group was apparently not essential for enzyme activity. EPNP was found to inactivate other gastric proteases: human gastricsin, human pepsin, and bovine rennin. The stoichiometry of the reaction in human gastricsin and pepsin was identical with that in porcine pepsin.