Challenging a paradigm: Theoretical calculations of the protonation state of the Cys25‐His159 catalytic diad in free papain

A central mechanistic paradigm of cysteine proteases is that the His–Cys catalytic diad forms an ion-pair NH(+)/S(−) already in the catalytically active free enzyme. Most molecular modeling studies of cysteine proteases refer to this paradigm as their starting point. Nevertheless, several recent kinetics and X-ray crystallography studies of viral and bacterial cysteine proteases depart from the ion-pair mechanism, suggesting general base catalysis. We challenge the postulate of the ion-pair formation in free papain. Applying our QM/SCRF(VS) molecular modeling approach, we analyzed all protonation states of the catalytic diad in free papain and its SMe derivative, comparing the predicted and experimental pKa data. We conclude that the His–Cys catalytic diad in free papain is fully protonated, NH(+)/SH. The experimental pKa = 8.62 of His159 imidazole in free papain, obtained by NMR-controlled titration and originally interpreted as the NH(+)/S(−) ⇌ N/S(−) equation image equilibrium, is now assigned to the NH(+)/SH ⇌ N/SHequation image equilibrium.