The contribution of intermolecular hydrogen bonding to the kinetic specificity of papain

The binding of substrates to the active site of papain is thought to involve, among other things, intermolecular P1NH-OC(Asp158) and P2NH-OC(Gly66) hydrogen bonding. In this study the contribution of these two putative hydrogen bonds to the interaction specificity of papain was measured for pairs of ligands in which the amide NH in question was either intact or replaced by an ester O linkage. The probe ligands investigated comprised substrates (peptidyl p-nitroanilides), substrate-like transition state analog inhibitors (peptidyl nitriles) and substrate-like affinity labeling agents (peptidyl Michael acceptors). Observed differences in interaction energies (delta delta G(obs) or delta delta G(obs) not equal to) for amide/ester ligand pairs indicated an apparent specificity energy of 2.1-2.6 kcal/mol for the P2NH-OC(Gly66) bond. For the P1NH-OC(Asp158) bond delta delta G(obs) was approx. 1.0 kcal/mol for dipeptidyl ligands but close to zero for ligands lacking a P2NH donor. These specificity energies are comparable to values reported for other enzyme-ligand systems. However, the dependence of delta delta G(obs) for the P1NH-OC(Asp158) bond on the presence of a P2NH donor suggests that these two hydrogen bonds may interact cooperatively in ligand binding. A thermodynamic cycle approach was used to relate delta delta G(obs) to actual hydrogen bond strengths and other aspects of enzyme-ligand and solvent-ligand interactions.