Journal of Biological Chemistry · 1990 · 199 citations · 13 references
Life SciencesBiochemistryMolecular BiologyEnzymologyMolecular VirologyVirologyAmino Acid PropertiesEnzyme CharacterizationEnzyme FunctionEnzyme InhibitionEnzyme KineticsProteolytic EnzymesIonic StrengthIonic Strength DependenceIonic Strength EffectsProtease Cleavage SpecificityProtease KineticsSpectrophotometric Enzyme AssaysViral Protease InhibitionViral Protease Specificity
By replacement of the P1' residue in a capsid/nucleocapsid cleavage site mimic with 4-NO2-phenylalanine (Nph), an excellent chromogenic substrate, Lys-Ala-Arg-Val-Leu*Nph-Glu-Ala-Met, for HIV-1 proteinase (kappa cat = 20 s-1, Km = 22 microM) has been prepared. Substitution of the Leu residue in P1 with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (kappa cat and kappa cat/Km) determined at different pH values, whereas peptides containing Ile or Val in P1 were hydrolyzed extremely slowly. The spectrophotometric assay has been used to characterize the proteinase further with respect to pH dependence, ionic strength dependence, and the effect of competitive inhibitors of various types.
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