Effect of pH and nonphysiological salt concentrations on human immunodeficiency virus-1 protease dimerization.
Human immunodeficiency virus-1 (HIV-1) protease is catalytically active 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 structure and function of the enzyme. Enzymatic activity was measured by following the hydrolysis of a fluorogenic substrate. Dissociation of the dimer into its subunits was monitored by gel filtration, while conformational changes in the enzyme were probed by measurements of intrinsic tryptophan fluorescence. Mg2+ ions were capable of dissociating the dimeric enzyme with a concomitant red shift and increase in quantum yield of the tryptophan fluorescence, indicating increased accessibility of tryptophan to the aqueous environment. These structural changes also were associated with a loss of catalytic activity which was insensitive to substrate concentration, consistent with noncompetitive inhibition. Both structural and functional changes could be attributed to binding of Mg2+ ions to a site with an apparent dissociation constant of approximately 2 M. In contrast, increasing concentrations of Na ions up to 5 M were without effect. Increasing pH had similar effects on HIV-1 protease as increasing Mg2+ ions concentration, with concomitant dissociation into subunits, increase in quantum yield and red shift in tryptophan fluorescence, and loss in catalytic activity. The apparent pKa for these structural and functional transitions was 6.95 +/- 0.08. This value is consistent with that of an aspartic acid residue with an anomalously high pKa, which has been implicated in the catalytic activity of HIV-1 protease.
Department of Medicine, University of Missouri, Columbia 65212.
S C
SC
Tyagi
S R
SR
Simon
C A
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Carter
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004926:Escherichia coli; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology
D002413:Cations, Divalent; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D012965:Sodium Chloride; D016333:HIV Protease; D008274:Magnesium