Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate.

Reaction mechanisms and rate constants of the acylation reaction of the hepatitis C virus (HCV) NS3/NS4A serine protease with the NS4B/5A natural substrate were studied using SCC-DFTB/MM (self-consistent charge density functional tight binding/molecular mechanics) and EA-VTST/MT (ensemble-averaged variational transition state theory/multidimensional tunneling) methods, considering the isotope effect (H/D). This reaction is crucial in the HCV life cycle. The reaction follows an essentially concerted mechanism. Although two elementary steps are involved, no intermediate step has been found between them. Thus, the proposed general two-step serine protease acylation mechanism, which includes a tetrahedral intermediate, does not occur here. This finding aligns with our studies on another natural substrate (NS5A/5B), indicating a greater variety in mechanism than previously expected. Tunneling and recrossing play an intermediate role; the activation free energy barriers are in good agreement with the experimental value, and the kinetic isotope effect (k(H)/k(D)) is somewhat larger than one (1.3). The rate constant value is not reproduced due to the exponential dependence of the rate constant on the activation free energy.