Kinetic assay for HIV proteinase subunit dissociation.
The kinetics and thermodynamics of the monomer--dimer equilibrium for HIV-1 proteinase are investigated in a concentration jump experiment, at a concentration of the substrate that is substantially lower than the Michaelis constant. Under these conditions the substrate-induced stabilization of the active dimer is suppressed, and the integral rate equation can be obtained in a closed form. Both the monomer--dimer bimolecular association rate constant and the corresponding equilibrium dissociation constant are obtained directly by nonlinear regression analysis of the reaction time-course. In buffers of low ionic strength and in the absence of external ligands (substrates and inhibitors), the equilibrium dissociation constant at 37 degrees C is 440 +/- 52 nM, a value significantly higher than previous estimates obtained at a comparatively high concentration of substrates.
Kuzmic
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University of Wisconsin, School of Pharmacy, Madison 53706.
D000595:Amino Acid Sequence; D016333:HIV Protease / Q000032:analysis / Q000737:chemistry*; D015497:HIV-1 / Q000201:enzymology*; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D012044:Regression Analysis; D013816:Thermodynamics
D046911:Macromolecular Substances; D010455:Peptides; D016333:HIV Protease