Reversible binding of peptide aldehydes to papain. Structure-activity relationships

Abstract

The hydration of eleven peptide and hippuryl aldehydes has been measured as a function of temperature by means of NMR spectroscopy. In all cases the aldehydes were strongly hydrated (i.e., 90–95%) in aqueous solution. Dehydration of the hydrates was strongly endothermic, but this was partly offset by a positive entropy for dehydration. The binding of the aldehydes to papain was measured by fluorescence titration, and from these data dissociation constants for the hemithioacetal enzyme adducts were derived. Binding of N-Ac-l-PheNHCH2CHO (1) was particularly tight (Kd,corr = 0.00043 μM) whereas that of its d-enantiomer (2) was 300-fold weaker (Kd,corr = 0.129 μM). The binding constants of the eleven aldehydes correlated with those for the reversible covalent binding of the analogous nitriles according to the equation log Kd(CHO) = −2.687 + 1.016 log Kd,corr(CN) (r = 0.99), lending support to previous suggestions that both peptide aldehydes and peptide nitriles behave as transition-state- or reactive intermediate analogs for papain. This finding is particularly striking in view of the obvious differences in hybridization (sp2 vs. sp3) and geometry (trigonal vs. tetrahedral) at the reactive P1 carbon center in their covalent adduct forms (thioimidate ester vs. hemithioacetal, respectively). A model for the binding of substrates, their transition states and analogs thereof is proposed. A key feature of the model is an obligatory covalent (or developing covalent) interaction between Cys-25-SH and the carbonyl or equivalent carbon of P1, augmented by intermolecular P1NHOC(Asp-158), P2COHN(Gly-66) and P2NHOC(Gly-66) hydrogen bonds and a hydrophobic P2S2 interaction. The latter three interactions are optimum or nearly optimum when P2 is a hydrophobic l-amino acid with an N-acyl substituent. Data presented suggest that hippuryl derivatives are relatively non-specific substrates or inhibitors for papain and, consequently, are of diminished value as probes for binding and catalytic studies.