EFFECT OF PH AND NONPHYSIOLOGICAL SALT CONCENTRATIONS ON HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE DIMERIZATION

Human immunodeficiency virus-1 (HIV-1) protease is catalytically activ e as a dimer of identical subunits that associate through noncovalent interactions. To investigate the forces stabilizing HIV-1 protease in its active form, we have studied the effects of pH and salts on struct ure and function of the enzyme. Enzymatic activity was measured by fol lowing the hydrolysis of a fluorogenic substrate. Dissociation of the dimer into its subunits was monitored by gel filtration, while conform ational changes in the enzyme were probed by measurements of intrinsic tryptophan fluorescence. Mg2+ ions were capable of dissociating the d imeric enzyme with a concomitant red shift and increase in quantum yie ld of the tryptophan fluorescence, indicating increased accessibility of tryptophan to the aqueous environment. These structural changes als o were associated with a loss of catalytic activity which was insensit ive to substrate concentration, consistent with noncompetitive inhibit ion. Both structural and functional changes could be attributed to bin ding of Mg2+ ions to a site with an apparent dissociation constant of similar to 2 M. In contrast, increasing concentrations of Na ions up t o 5 M were without effect. Increasing pH had similar effects on HIV-1 protease as increasing Mg2+ ions concentration, with concomitant disso ciation into subunits, increase in quantum yield and red shift in tryp tophan fluorescence, and loss in catalytic activity. The apparent pK(a ) for these structural and functional transitions was 6.95 +/- 0.08. T his value is consistent with that of an aspartic acid residue with an anomalously high pK(a), which has been implicated in the catalytic act ivity of HIV-1 protease.