Studies on the Extended Active Site of Papain

Abstract A series of papain substrates of the type A-Val-Glu-Leu-Gly has been synthesized, in which the Glu-Leu bond is the only one cleaved by the enzyme under the conditions of these studies, and in which the nature of the A group has been varied. Estimates have been made of the apparent Km and kcat in the hydrolysis of these substrates, and the results are consistent with the view that papain possesses an extended active site. To examine the effect of changes in the A group on the interaction of papain with oligopeptides, the 6-(N-methylanilino)-2-naphthalenesulfonyl (mansyl, Mns) group was introduced as a fluorescent probe at the NH2 terminus (A = Mns, Mns-Gly, Mns-Gly-Gly). Steady state fluorescence measurements showed that neither active papain nor papain inactivated by blockage of the active site sulfhydryl group has appreciable intrinsic affinity for the mansyl group, but that the mansyl group of compounds such as Mns-Gly-Val-Glu-Leu-Gly is drawn, by peptide-protein interaction, into an environment of lower dynamic polarity. This effect was most marked with mercuripapain, and can be reversed by the addition of a specific substrate (benzoyl-l-arginine ethyl ester), suggesting that blockage of the active site sulfhydryl group does not prevent the binding of peptides at the extended active site of papain.