An ab Initio Quantum Mechanical Model for the Catalytic Mechanism of HIV-1 Protease Hsing Lee,* ,† Thomas A. Darden, † and Lee G. Pedersen ‡ Contribution from the Laboratory of QuantitatiVe and Computational Biology, National Institute of EnVironmental Health Science, Research Triangle Park, North Carolina 27709, and Department of Chemistry, UniVersity of North Carolina, Chapel Hill, North Carolina 27599-3290 ReceiVed April 25, 1995. ReVised Manuscript ReceiVed January 19, 1996 X Abstract: The catalytic mechanism of the HIV-1 protease (HIV-PR) is studied through ab initio theoretical model calculations. This model consists of a formate/formic acid pair, a structurally important water molecule, and a formamide molecule. The proposed catalytic mechanism is composed of five steps, two of which are transition states separated by a third step (an intermediate state). The remaining two steps are related to product release. The overall forward hydrolysis reaction barrier is approximately 22 kcal/mol, with a reverse hydrolysis barrier of approximately 34 kcal/mol at the RHF/6-31G* level. The second transition state is related to a nucleophilic attack of the water molecule on the carbon atom of the substrate scissile bond, and is essential for the collapse of the substrate. That the transition state structures of HIV-PR have not been identified makes a theoretical study of this kind particularly valuable for understanding the HIV-PR mechanism. Introduction HIV type 1 protease (HIV-PR) is a 99-amino acid protein that is crucial for the maturation of the HIV virion. HIV-PR catalyzes its own release from the poly-protein Pr160 gag-pol , the protein product of HIV-DNA, by hydrolysis of certain peptide bonds. Once free, the PR catalyzes a series of hydrolytic cleavages resulting in the final proteins of the matured form of the HIV virus. 1 It has been found 2 that effective inhibition of HIV-PR leads to production of a noninfectious form of the virus. These considerations make HIV-PR an attractive therapeutic target. The active site triad Asp-Thr-Gly (residues 25-27) of HIV- PR is characteristic of aspartic proteases, a well-known family of proteases. The active form of HIV-PR is dimeric, the structural details of which became clear after the determination of the crystal structure of a synthetic HIV-PR at a resolution of 2.8 Å by Wlodawer et al. 3 It follows from this structure that the two side chains of Asp-25 and Asp-25′ of the active site are planar with a structural water molecule bound to both. Meek and co-workers 4 presented evidence from kinetic studies that at physiological conditions one of the carboxylate groups of the aspartate side chains is protonated while the other is not. The water molecule has been postulated to carry out the nucleophilic attack on the C atom of the substrate scissile bond. The role for the rest of the active site triad remains unclear. In the X-ray crystallographic structure of HIV-PR, the N atoms of Gly-27 and Gly-27′ are in a position to form H-bonds to the O δ atoms of D25/D25′. However, experiments to date appear to indicate that these glycine residues do not participate in the catalysis directly. It may be reasonable to assume that glycine 27 and glycine 27′ are responsible for maintaining the planarity of the D25/D25′ motif. It appears from the isotope kinetic experiments 4 that two major components are involved in the HIV-1 PR catalysis. First, proton transfers involving both the scissile nitrogen and carbon atoms of the substrate are involved during the hydrolysis. Second, an additional water molecule participates in the product release. While these factors are inconsistent with some earlier mechanisms that have been proposed, 8 two proposals appear to be the most plausible. The essence of the proposal by Meek and co-workers, 4b which we have not evaluated in this study, is the formation of an amide hydrate intermediate involving the lytic water molecule and several concerted proton transfers. This proposal will ultimately be interesting to study with theoretical calculations. The current theoretical study has been formulated to follow closely the proposal by Jaskolski et al. 9 The original assump- tions of this proposal are the following: (1) A concerted electrophilic attack of a proton of the active site on the target nitrogen atom of the substrate scissile bond together with a nucleophilic attack of the water molecule on the C atom of the substrate peptide bond start the reaction and lead to a transition state. 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