title: Activity and dimerization of human immunodeficiency virus protease as a function of solvent composition and enzyme concentration.
abstract: The activity of human immunodeficiency virus 1 (HIV-1) protease has been examined as a function of solvent composition, incubation time, and enzyme concentration at 37 degrees C in the pH 4.5-5.5 range. Glycerol and dimethyl sulfoxide inhibit the enzyme, while polyethylene glycol and bovine serum albumin activate the enzyme. When incubated at a concentration of 50-200 nM, the activity of the protease decreases irreversibly with an apparent first-order rate constant of 4-9 x 10(-3) min-1. The presence of 0.1% (w/v) polyethylene glycol or bovine serum albumin in the reaction buffer dramatically stabilizes enzyme activity. In the absence of prolonged incubation of the enzyme at submicromolar concentration, the specific activity of HIV-1 protease in buffers of either high or low ionic strength is constant over the enzyme concentration range of 0.25-5 nM, indicating that dissociation of the dimeric protease, if occurring, can only be governed by a picomolar dissociation constant. Similarly, the variation of the specific activity of HIV-2 protease over the enzyme concentration of 4-85 nM is consistent only with a dimer dissociation constant of less than 10 nM. We conclude that: 1) the assumption of a nondissociating HIV-1 protease is a valid one for kinetic studies of tight-binding inhibitors where nanomolar concentrations of the enzymes are employed; 2) stock protease solutions of submicromolar concentration in the absence of activity-stabilizing compounds may lead to erroneous kinetic data and complicate mechanistic interpretations.
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: S P
authors.initials: SP
authors.lastname: Jordan
authors.forename: J
authors.initials: J
authors.lastname: Zugay
authors.forename: P L
authors.initials: PL
authors.lastname: Darke
authors.forename: L C
authors.initials: LC
authors.lastname: Kuo
mesh_terms: D000595:Amino Acid Sequence; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D016333:HIV Protease / Q000378:metabolism*; D017320:HIV Protease Inhibitors; D015497:HIV-1 / Q000201:enzymology*; D015498:HIV-2 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D012997:Solvents
chemical_list: D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D012997:Solvents; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D016333:HIV Protease; D005990:Glycerol; D004121:Dimethyl Sulfoxide