Effects of ligand homologation and ligand reactivity on the apparent kinetic specificity of papain.

Papain, a prototype cysteine proteinase, shows a pronounced kinetic preference for substrates and inhibitors based on the Ac-L-Phe-Gly-structural motif. Replacing the L-Phe at position P2 with D-Phe, or with a less hydrophobic residue such as Leu or Met, results in decreases of substrate or inhibitory activity of up to 400-fold. In this study we examined the effect of homologating the P1 glycine moiety to beta-alanine in the context of specific ester and amide substrates, peptidyl nitrile and -aldehyde transition state analog inhibitors, and peptidyl Michael acceptors as irreversible affinity labels. Papain discriminates extremely strongly (i.e., from 1000-fold to > or = 29,000-fold) against the 'homologs' based on beta-alanine at P1 compared to 'analogs' based on glycine at P1. However, with highly reactive ligands such as p-nitrophenyl esters, homolog/analog discrimination is greatly reduced (i.e., < or = 10-fold). These observations are interpreted in terms of (1) cooperativity between several non-covalent enzyme-ligand interactions and the covalent interaction of the ligand P1 moiety with Cys-25 of papain, (2) the decreased ability of homologs to utilize these cooperative interactions optimally because of their extended size, and (3) a decrease in the importance of the cooperative interactions as the intrinsic chemical reactivity of the ligand increases. Some implications of this analog vs. homolog discrimination for peptidyl disulfide and peptidyl chloromethane probes of protease specificity and mechanism are discussed.