DETERMINATION OF KINETIC RATE CONSTANTS FOR THE BINDING OF INHIBITORSTO HIV-1 PROTEASE AND FOR THE ASSOCIATION AND DISSOCIATION OF ACTIVE HOMODIMER

Association and dissociation rate constants for a competitive inhibito r of HIV-1 protease were determined by a novel method employing a pair of integrated rate equations. This method, termed the paired progress curve method, is both rapid and reproducible. Progress curves, taken at a single concentration of inhibitor, are analyzed simultaneously to determine association and dissociation rate constants, the concentrat ion of active sites, and the catalytic rate constant. The method is ap plied to BILA 398, a compound for which the cocrystal structure with H IV-2 protease has been reported recently [Tong, L., et al. (1993) Proc . Natl. Acad. Sci. U.S.A. 90, 8387-8391]. This compound exhibited an a ssociation constant of 1.6 x 10(7) M(-1) s(-1) and a dissociation cons tant of 1.0 x 10(-4) s(-1) corresponding to a binding affinity constan t of 6.4 x 10(-12) M. During the course of the analysis, nonlinearity was observed in control reactions containing enzyme and substrate only . This was subsequently shown to be due to a reversible inactivation p rocess resulting from enzyme dilution. Integrated rate equations were developed on the basis of the dissociation of active dimeric enzyme du ring dilution and a reassociation of dilute monomers following the add ition of substrate. The equations were modeled to the data, yielding a dissociation constant of 1.9 x 10(-3) s(-1) and an association consta nt of 9.2 X 10(5) M(-1) s(-1) for the monomer-dimer interconversion pr ocess. This corresponds to an equilibrium constant of 4 x 10(-9) M for the dimerization of HIV-1 protease.