The pKa of the catalytic His57 N(epsilon)H in the tetrahedral complex (TC) of chymotrypsin with trifluoromethyl ketone inhibitors is 4-5 units higher relative to the free enzyme (FE). Such stable TC's, formed with transition state (TS) analog inhibitors, are topologically similar to the catalytic TS. Thus, analysis of this pKa shift may shed light on the role of water solvation in the general base catalysis by histidine. We applied our QM/SCRF(VS) approach to study this shift. The method enables explicit quantum mechanical DFT calculations of large molecular clusters that simulate chemical reactions at the active site (AS) of water solvated enzymes. We derived an analytical expression for the pKa dependence on the degree of water exposure of the ionizable group, and on the total charge in the enzyme AS, Q(A) and Q(B), when the target ionizable functional group (His57 in this study) is in the acidic (A) and basic (B) forms, respectively. Q2(B) > Q2(A) both in the FE and in the TC of chymotrypsin. Therefore, water solvation decreases the relative stability of the protonated histidine in both. Ligand binding reduces the degree of water solvation of the imidazole ring, and consequently elevates the histidine pKa. Thus, the binding of the ligand plays a triggering role that switches on the cascade of catalytic reactions in serine proteases.

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.

Various mechanisms for the reversible formation of a covalent tetrahedral complex (TC) between papain and peptidyl aldehyde inhibitors were simulated by DFT calculations, applying the quantum mechanical/self consistent reaction field (virtual solvent) [QM/SCRF(VS)] approach. Only one mechanism correlates with the experimental kinetic data. The His-Cys catalytic diad is in an N/SH protonation state in the noncovalent papain-aldehyde Michaelis complex. His159 functions as a general base catalyst, abstracting a proton from the Cys25, whereas the activated thiolate synchronously attacks the inhibitor's carbonyl group. The final product of papain inhibition is the protonated neutral form of the hemithioacetal TC(OH), in agreement with experimental data. The predicted activation barrier g enz≠ = 5.2 kcal mol⁻¹ is close to the experimental value of 6.9 kcal mol⁻¹. An interpretation of the experimentally observed slow binding effect for peptidyl aldehyde inhibitors is presented. The calculated g cat≠ is much lower than the rate determining activation barrier of hemithioacetal formation in water, g w≠, in agreement with the concept that the preorganized electrostatic environment in the enzyme active site is the driving force of enzyme catalysis. We have rationalized the origin of the acidic and basic pK(a)'s on the k₂/K(S) versus pH bell-shaped profile of papain inhibition by peptidyl aldehydes.

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 hitherto unsolved problem of the origin of the unusually high nitrogen inversion-rotation (NIR) barriers in 7-azabicyclo[2.2.1]heptanes (the bicyclic effect) was examined using the natural bond orbital (NBO) approach. Reinvestigating the NIR barrier for tropane by DNMR, we found that NIR barriers increase smoothly on going from nitrogen-bridged bicyclic systems of a larger ring size to the smaller ring homologous systems. The experimental NIR barriers are reproduced with good accuracy using the MP2/6-31G level of theory. The NBO analysis for these and other azabicycles led to the conclusion that the height of these barriers is mostly determined by the energy of the sigma-orbitals of the C(alpha)(-)C(beta) bonds as well as the nitrogen lone pair. Thus, the bicyclic effect is actually an extreme case of a common C(alpha-)N-C(alpha) tripyramid geometry-NIR barrier dependence for N-bridged bicyclic amines. By establishing the rate-determining role of the C(alpha-)N-C(alpha) tripyramid fragment for NIR, we have derived the first sufficiently accurate quantitative correlations amine geometry-NIR barrier for monocyclic as well as bicyclic N-H and N-Me amines (i.e., for an amine set which also includes the bicyclic effect systems).