Reversible covalent binding of peptide nitriles to papain.

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of fluorescence titration. The binding constants generally parallel the kinetic specificity constants (kcat/Km) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of delta H vs. T delta S resulted in two parallel lines, one for 'specific' nitriles (i.e., N-Ac-L-aa-NHCH2CN; aa = Phe, Leu, Met) and the other for 'non-specific' nitriles (e.g., N-Ac-D-Phe-NHCH2CN, PhCH2CH2CONHCH2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of Kd values (0.27 microM-490 microM), the solvent deuterium isotope effect on binding (Kd(H2O)/Kd(D2O) = DKd) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.