Determination of kinetic rate constants for the binding of inhibitors to HIV-1 protease and for the association and dissociation of active homodimer.

Important note: Information in this article was accurate in 1995. The state of the art may have changed since the publication date.

Determination of kinetic rate constants for the binding of inhibitors to HIV-1 protease and for the association and dissociation of active homodimer.

Biochemistry. 1994 Oct 18;33(41):12527-34. Unique Identifier : AIDSLINE MED/95001975
Pargellis CA; Morelock MM; Graham ET; Kinkade P; Pav S; Lubbe K; Lamarre D; Anderson PC; Department of Biochemistry, Boehringer Ingelheim Pharmaceuticals,; Inc., Ridgefield, Connecticut 06877.

Abstract: Association and dissociation rate constants for a competitive inhibitor 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 concentration of active sites, and the catalytic rate constant. The method is applied to BILA 398, a compound for which the cocrystal structure with HIV-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 association constant of 1.6 x 10(7) M-1 s-1 and a dissociation constant of 1.0 x 10(-4) s-1 corresponding to a binding affinity constant 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 process resulting from enzyme dilution. Integrated rate equations were developed on the basis of the dissociation of active dimeric enzyme during dilution and a reassociation of dilute monomers following the addition of substrate. The equations were modeled to the data, yielding a dissociation constant of 1.9 x 10(-3) s-1 and an association constant of 9.2 x 10(5) M-1 s-1 for the monomer-dimer interconversion process. This corresponds to an equilibrium constant of 4 x 10(-9) M for the dimerization of HIV-1 protease.

Keywords: Binding, Competitive HIV Protease/CHEMISTRY/*METABOLISM HIV Protease Inhibitors/*METABOLISM HIV-1/*ENZYMOLOGY Kinetics Macromolecular Systems Regression Analysis JOURNAL ARTICLE
950130
M9510807

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