Figure 2. Kinetic analysis of TDPX2 with TryX and cumene hydroperoxide as substrate.

(A) The initial velocities were determined with varied cumene hydroperoxide concentrations and different TryX concentrations (■, 12.5 μM; □, 10 μM; ●, 7.5 μM; ○, 5 μM). The initial velocities for each individual data set were initially fitted individually to the Michaelis–Menten equation (broken line) by non-linear regression using GraFit and subsequently the entire data set was globally fitted to the equation describing a Ping Pong mechanism (continuous line). (B) The reciprocal V^app_max data, calculated for each TryX concentration data set, were plotted against the reciprocal TryX concentrations. The reciprocal intercept represents the reciprocal V_max value and the reciprocal slope the rate constant k₂ for TryX. (C) The reciprocal initial velocities are plotted against the reciprocal cumene hydroperoxide concentrations. This Figure shows the accuracy of the two fits transformed as a Lineweaver–Burk plot (continuous line, global fit; broken line, individual fits for each TryX concentration). The slope represents the reciprocal rate constant, k₁, for cumene hydroperoxide. Kinetic constants are reported in Table 1.