We studied the role of D-158 in papain-like cysteine proteinases by using subtilisin Carlsberg, and its chemically modified analog thiolsubtilisin, by applying the proton inventory (PI) method and also by taking into account the pH profiles of the k(cat)/K-m parameter. In the case of thiolsubtilisin, we estimated large inverse solvent isotope effects for k(cat)/K-m, as in papain, whereas for subtilisin we found "dome-shaped" PI, suggesting a completely different mechanism. Finally, the kinetic behavior of thiolsubtilisin presented similarities as well as differences, compared to papain, suggesting a possible role for D-158 as part of a catalytic triad in papain-like cysteine proteinases.