Dissociation and association of the HIV-1 protease dimer subunits: equilibria and rates.

Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.

Dissociation and association of the HIV-1 protease dimer subunits: equilibria and rates.

Biochemistry. 1994 Jan 11;33(1):98-105. Unique Identifier : AIDSLINE MED/94114519
Darke PL; Jordan SP; Hall DL; Zugay JA; Shafer JA; Kuo LC; Department of Biological Chemistry, Merck Research Laboratories,; West Point, Pennsylvania 19486.

Abstract: The kinetics and equilibrium properties were investigated for the interconversion between the active dimer of human immunodeficiency virus 1 (HIV-1) protease and its inactive monomeric subunits. The equilibrium dissociation constant (Kd) of the dimeric protease as well as the monomer association rate were obtained by monitoring the fluorescence change of an active-site-directed fluorescent probe (L-737244) upon its binding to the protease. The Kd of the HIV-1 protease is strongly pH dependent. At pH 5.5 where the enzyme is most active catalytically, the extrapolated values of Kd are 0.75 and 3.4 nM at 30 and 37 degrees C, respectively. The rate constant for HIV-1 monomer association, approximately 4 x 10(5) M-1 s-1, is within the range commonly observed for protein-protein interactions. Dimer dissociation was further scrutinized in the presence of an inactive, point mutant form of the enzyme. As a result of subunit exchange between the native and mutant enzymes and the formation of an inactive heterodimer, there was a time-dependent decrease in the activity of the native protease. Enzyme activity could be reinstated with the addition of an active-site-directed inhibitor (L-365862) which selectively binds active dimers. The rate of dimer dissociation was found to also decrease with pH. At pH 5.5 and 30 degrees C, the half-life for subunit dissociation is about 0.5 h. The slow dissociation, coupled with the high stability for dimer association, attests to the importance of allowing sufficient time for dimer-monomer equilibration in kinetic assays in order to avoid reaching erroneous conclusions in studies of dimer dissociation.

Keywords: Amino Acid Sequence Hydrogen-Ion Concentration HIV Protease/*CHEMISTRY/*METABOLISM HIV Protease Inhibitors/METABOLISM/PHARMACOLOGY HIV-1/*ENZYMOLOGY Kinetics Macromolecular Systems Mathematics Models, Theoretical Molecular Sequence Data Oligopeptides/CHEMICAL SYNTHESIS/METABOLISM Pyrrolidinones/METABOLISM/PHARMACOLOGY Thermodynamics Time Factors Valine/ANALOGS & DERIVATIVES/METABOLISM/PHARMACOLOGY JOURNAL ARTICLE
940430
M9440308

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