Molecular mechanism of inhibition of cysteine proteinases by their protein inhibitors: kinetic studies with natural and recombinant variants of cystatins and stefins.

Natural and recombinant variants of the cysteine proteinase inhibitors chicken cystatin and human stefin B were characterized by determination of their inhibition constants for papain, actinidin and human cathepsins B and H. The individual contributions of the three contact regions to the binding energy of the chicken cystatin-papain complex were calculated as 36% for the N-terminal segment, 51% for the first and 13% for the second hairpin loop. Removal of the N-terminal contact region of chicken cystatin resulted in a 10000-fold lower affinity for papain. In contrast, stefin B remained a tight-binding inhibitor of papain and actinidin without its N-terminal segment. Affinity of stefin B for papain was only slightly affected by exchange of the residue predicted to bind in the S2 subsite of papain. The essential contribution of the first hairpin loop to inhibitor binding was confirmed by the 240-fold lower affinity for papain of a Val48----Asp mutant of stefin B. Inhibition of cathepsin B by stefins A and B is slow-binding. Binding of stefin B, not of stefin A, follows a two-step mechanism involving a slow isomerisation of the enzyme-inhibitor complex.