Theoretical Study of the Free Energy Surface and Kinetics of the Hepatitis C Virus NS3/NS4A Serine Protease Reaction with the NS5A/5B Substrate. Does the Generally Accepted Tetrahedral Intermediate Really Exist?

The self-consistent charge density functional tight binding/molecular mechanics (SCC-DFTB/MM) and ensemble averaged variational transition state theory/multidimensional tunneling (EA-VTST/MT) methods have been employed to investigate the reaction mechanism and to calculate the rate constant of the NS3/NS4A + NS5A/5B acylation reaction. This reaction belongs to the vital cycle of the hepatitis C virus once it infects the human cell. A concerted reaction mechanism, with a single transition state in which the tetrahedral geometry has already been adopted and the peptide bond is starting to break, has been determined. This reaction supposes an example where the proposed general two-step serine protease acylation reaction mechanism does not occur, being related to the fact that the enzyme is particularly efficient for the NS5A/5B substrate. The transition state characterized here for the acylation reaction can be a good initial structure in the reach of NS3/NS4A inhibitors based on TS analogues. On the other hand, the calculated and experimental phenomenological free energy barriers only differ by 2.3 kcal mol–1, although this leads to a significant discrepancy between calculated and experimental. The rest of the calculated kinetic parameters, such as the kinetic isotopic effect (H/D), tunneling, and the recrossing contributions to reactivity, agree with the expected behavior for the studied reaction.