Cysteine proteases: the S2P2 hydrogen bond is more important for catalysis than is the analogous S1P1 bond.

High hydrophobicity of the second amino acid N-terminal to the scissile bond (P2 residue) is generally considered to be the major factor in the specificity of the substrates for cysteine proteases of the papain family. To examine the catalytic contribution of the S2P2 hydrogen bond apparent from X-ray crystallographic studies, the kinetics of Z-Phe-Gly-OEt and its thiono derivative were compared. The thiono compound contains a sulfur atom in place of the carbonyl oxygen of the phenylalanine residue. It was found that the specificity rate constants for the reactions of the thiono substrate with various cysteine proteases are lower by 2-3 orders of magnitude as compared to the corresponding rate constants for the oxo substrate. This remarkable effect is not expected in the light of previous studies indicating that the change from oxygen to sulfur in the P1 residue was without an appreciable effect. The results are interpreted in terms of a distorted binding of the thiono substrate.