Ab initio evaluation of the free energy surfaces for the general base/acidcatalyzed thiolysis of formamide and the hydrolysis of methyl thiolformate: A reference solution reaction for studies of cysteine proteases

Although the catalytic reaction of cysteine proteases is a process of major importance, we do not have a quantitative understanding of the relevant en ergetics. The present work takes a crucial step in this direction and deter mines the free energy surface for the corresponding reference solution reac tion. The calculations involve the evaluation of the potentials of the mean force for ammonia-, histidine, and water-assisted reactions of thiomethano l with formamide in aqueous solution, as well as for the hydrolysis of the resulting thiolester. These calculations were carried out using the quantum mechanical B3LYP/AUG-cc-pVDZ//HF/6-31G* method and the Langevin dipoles so lvation model. The calculations involve self-consistent evaluation of the s olute charges in solution as well as mapping the solution free energy surfa ce (rather than the gas-phase surface). The amide thiolysis (acylation step ) was found to have a stepwise character with equal activation barriers of 24 kcal/mol for histidine-assisted nucleophilic attack on amide, and the el imination of NH3 from the resulting tetrahedral intermediate. This mechanis m is quite different than the mechanism suggested by previous gasphase theo retical studies. The subsequent hydrolysis of methyl thiolformate (deacylat ion step) also has stepwise character, with H2O attack in the histidine-ass isted reaction characterized by a barrier of 26 kcal/mol. The anionic tetra hedral intermediate formed by this attack decomposes into formic acid and t hiolate anion with the activation free energy of 25 kcal/mol. Our calculati ons lead to the conclusion that the active sites of cysteine proteases must provide substantial stabilization to four different transition state struc tures.