HIV-1 Protease:  Characterization of a Catalytically Competent Enzyme−Substrate Intermediate

The steady-state and pre-steady-state kinetic parameters for the interaction of E with the fluorogenic substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg-NH2 were determined in 1.25 M NaCl, 0.1 M MES−TRIS at pH 6.0 at 25 °C. At low concentrations of enzyme, the values of the K m and k cat calculated from steady-state data were 2.1 μM and 7.4 s-1, respectively. At high concentrations of enzyme, the time-courses of fluorescence enhancement associated with catalysis were very dependent on the excitation wavelength used to monitor the reaction. Because the absorbance spectrum of the substrate overlapped the fluorescence emission spectrum of the enzyme, these abnormalities were attributed to fluorescence energy transfer between the enzyme and the substrate in an enzyme−substrate intermediate. The kinetic data collected with λex = 280 nm and λem > 435 nm were analyzed according to the following mechanism in which EX was the species with enhanced fluorescence relative to substrate or products: The values of the kinetic parameters with 1H2O as the solvent were K = 13 μM, k 2 = 150 s-1, k - 2 = 25 s-1, and k 3 = 11 s-1. The values of the kinetic parameters with 2H2O as the solvent were K = 13 μM, k 2 = 210 s-1, k - 2 = 12 s-1, and k 3 = 4.4 s-1. These values yielded solvent isotope effects of 2 on k cat and 0.9 on k cat/K m. From analysis of the complete time-course of the fluorescence change (λex = 280 nm and λem > 435 nm) during the course of substrate hydrolysis, the intermediate EX was determined to be 6.3-fold more fluorescent than the product, which, in turn, was 4.5-fold more fluorescent than ES or S. Rapid quench experiments with 2 N HCl as the quenching reagent confirmed that EX was a complex between enzyme and substrate. Consequently, the small burst in fluorescence observed when monitoring with λex = 340 nm (0.3 product equiv per enzyme equivalent) was attributed to the fluorescence change upon transfer of substrate from an aqueous environment to a nonaqueous environment in the enzyme. These results were consistent with carbon−nitrogen bond cleavage being the major contributor to k cat.