INHIBITION AND CATALYTIC MECHANISM OF HIV-1 ASPARTIC PROTEASE

The structure of the HIV-1 protease in complex with a pseudo-C2 symmet ric inhibitor, which contains a central difluoroketone motif, has been determined with X-ray diffraction data extending to 1.7 Angstrom reso lution. The electron density map clearly indicates that the inhibitor is bound in a symmetric fashion as the hydrated, or gemdiol, form of t he difluoroketone. Refinement of the complex reveals a unique, and alm ost symmetric, set of interactions between the geminal hydroxyl groups , the geminal fluorine atoms, and the active-site aspartate residues. Several hydrogen bonding patterns are consistent with that conformatio n. The lowest energy hydrogen disposition, as determined by semiempiri cal energy calculations, shows only one active site aspartate protonat ed. A comparison between the corresponding dihedral angles of the difl uorodiol core and those of a hydrated peptide bond analog, calculated nb-initio, shows that the inhibitor core is a mimic of a hydrated pept ide bond in a gauche conformation. The feasibility of an anti-gauche t ransition for a peptide bond after hydration is verified by extensive molecular dynamics simulations. The simulations suggest that rotation about the C-N scissile bond would readily occur after hydration and wo uld be driven by the optimization of the interactions of peptide side- chains with the enzyme. These results, together with the characterizat ion of a transition state leading to bond breakage via a concerted exc hange of two protons, suggest a proteolysis mechanism whereby only one active site aspartate is initially protonated. The steps of this mech anism are: asymmetric binding of the substrate; hydration of the pepti dic carbonyl by an active site water; proton translocation between the active site aspartate residues simultaneously with carbonyl hydration ; optimization of the binding of the entire substrate facilitated by t he flexible structure of the hydrated peptide bond, which, in turn, fo rces the hydrated peptide bond to assume a gauche conformation; simult aneous proton exchange whereby one hydroxyl donates a proton to the ch arged aspartate, and, at the same time, the nitrogen lone pair accepts a proton from the other aspartate; and, bond breakage and regeneratio n of the initial protonation state of the aspartate residues. (C) 1996 Academic Press Limited