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.