Inhibition of papain with 2-benzyl-3,4-epoxybutanoic acid esters. Mechanistic and stereochemical probe for cysteine protease catalysis.

ABSTRACT

Papain, a prototypic cysteine protease was inactivated by methyl and benzyl esters of (2S,3S)-2-benzyl-3,4-epoxybutanoic acid. On the other hand, methyl ester of (2S,3R)-2-benzyl-3,4-epoxybutanoic acid was shown to be a competitive inhibitor for the enzyme. It was inferred from the inactivation stereochemistry that in the papain catalytic reaction the nucleophilic attack of the side chain thioalkoxide of Cys-25 on the scissile peptide bond of substrates occurs in the 're' fashion. The papain inactivating potency of (2S,3S)-2-benzyl-3,4-epoxybutanoic acid methyl ester was enhanced over three-fold in a pH 8.0 solution compared with in the neutral solution. This together with our previous observation with alpha-chymotrypsin and the recent theoretical treatment of the enzymic reaction of papain, suggest that in the inactivation of papain by oxirane containing inhibitors, the oxirane does not need to be activated by prior protonation as thought previously. The oxirane ring is sufficiently labile that the unprotonated oxirane moiety can undergo an electrophilic reaction with the Cys-25 thiolate.