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

Abstract

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 pK a data. We conclude that the His -Cys catalytic diad in free papain is fully protonated, NH(+)/SH. The experimental pK a =8.62 of His159 imidazole in free papain, obtained by NMR controlled titratin and originally interpreated as the NH(+)/S(−) ⇌ N/S(−) equilibrium, is now assigned to the NH(+)/SH ⇌ N/SH equilibrium.

The mechanism of papain inhibition by peptidyl aldehydes

“…We recently demonstrated that in contrast to the paradigm, the His–Cys catalytic diad in free papain is fully protonated, NH(+)/SH 15. Our conclusion was supported by reproduction of the experimental p K a 's of His159 and the 1 H NMR chemical shifts of the CH ϵ proton of His159 16, 17…”

From Catalytic Mechanism to Rational Design of Reversible Covalent Inhibitors of Serine and Cysteine Hydrolases

“…[61,75] It is widely accepted that their catalytic dyad forms an imidazoliumÀthiolate ion pair NH + /S À in the free enzyme (FE). [87] We demonstrated that the pK a values of the catalytic residues were controlled by the cooperative effect of the AS polar local environment (preoriented dipoles) and the free energy of solvation of the partially water exposed target catalytic group in the AS. The His-ImH + functions as a general acid, facilitating leaving group departure from the tetrahedral intermediate to produce an acylÀenzyme intermediate.…”

“…By applying our QM/SCRF(VS) molecular modeling approach (see below for some details about the method) [83] to simulate the enzyme AS on a QM molecular cluster, [84][85][86] we analyzed all possible protonation states of the catalytic dyad in free papain (FE) and its derivative (SSMe), in which the catalytic thiol was blocked by methylthiolation ( Figure 2). [87] We demonstrated that the pK a values of the catalytic residues were controlled by the cooperative effect of the AS polar local environment (preoriented dipoles) and the free energy of solvation of the partially water exposed target catalytic group in the AS. Based on our modeling simulations of all possible protonation states of the catalytic dyad, calculated versus ex-perimental pK a values, we suggested a novel structure for the catalytic Cys25-SH-His159-ImH + dyad in free papain at neutral pH, in which both residues were protonated, in contrast to the ion-pair paradigm.…”

“…[47,51,83,87,106] Our interpretation of the catalytic mechanism of cysteine protease inhibition by petidyl aldehyde inhibitors demonstrates that the final thermodynamically stable product is a neutral covalent TC(OH) that principally differs from the anionic TC(O À ) species formed by inhibition of serine proteases. [47,51,83,87,106] Our interpretation of the catalytic mechanism of cysteine protease inhibition by petidyl aldehyde inhibitors demonstrates that the final thermodynamically stable product is a neutral covalent TC(OH) that principally differs from the anionic TC(O À ) species formed by inhibition of serine proteases.…”

“…We have elaborated the QM/SCRF(VS) approach for the theoretical exploration of enzymatic mechanisms and applied it to serine and cysteine hydrolases. [47,51,83,87,106] Our interpretation of the catalytic mechanism of cysteine protease inhibition by petidyl aldehyde inhibitors demonstrates that the final thermodynamically stable product is a neutral covalent TC(OH) that principally differs from the anionic TC(O À ) species formed by inhibition of serine proteases. The driving force of this phenomenon was theoretically explained as originating from the intrinsic difference in the electronic structure of thiol and oxygen nucleophiles.…”