FIGURE 5.
Effect of R212A, T224A, and R212A/T224A mutations in CYP3A4 on the kinetics of BEC binding. A–D, kinetics of BEC binding to the WT and R212A, T224A, and R212A/T224A mutants of CYP3A4, respectively. Solutions of 0.12, 0.25, 0.5, 1, 2, 4, 6, 12, 24, and 36 μm BEC (top to bottom traces, respectively) were mixed with 6 μm CYP3A4, and the low-to-high spin conversion was monitored at 417 nm. The kinetics were biphasic for the whole range of BEC concentrations studied. E and F, observed rate constants for the fast (kfast) and slow phases (kslow), respectively, are plotted versus BEC concentration. The rate constants calculated at saturating BEC are given in Table 2. G, effect of the BEC concentration on the relative percentage of the slow phase. The arrows indicate where the BEC and CYP3A4 concentrations are equal.