title: Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange.
abstract: The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.
authors.affiliation: Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: L J
authors.initials: LJ
authors.lastname: Hyland
authors.forename: T A
authors.initials: TA
authors.lastname: Tomaszek
authors.forename: G D
authors.initials: GD
authors.lastname: Roberts
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: V W
authors.initials: VW
authors.lastname: Magaard
authors.forename: H L
authors.initials: HL
authors.lastname: Bryan
authors.forename: S A
authors.initials: SA
authors.lastname: Fakhoury
authors.forename: M L
authors.initials: ML
authors.lastname: Moore
authors.forename: M D
authors.initials: MD
authors.lastname: Minnich
authors.forename: J S
authors.initials: JS
authors.lastname: Culp
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D016333:HIV Protease / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010103:Oxygen Isotopes
chemical_list: D009842:Oligopeptides; D010103:Oxygen Isotopes; D016333:HIV Protease