Figure 4.

Concentration dependence of αKG concentration on the observed rate of NADPH depletion in the neomorphic reaction (37 °C). The determined k_obs values were obtained from two different enzyme preparations to ensure reproducibility. The k_obs values resulting from each of the two enzyme preparations are distinguished by using either a circle or an × in the plots. The observed rate constants (k_obs) were calculated from the linear range of the slopes of plots of concentration versus time using GraphPad Prism software (GraphPad). These k_obs values were then fit to a hyperbolic equation to generate k_cat and K_m values, and the S.E. results from the deviance from these hyperbolic fits is indicated. K_m values and efficiency are in terms of [αKG]. Due to limits of detection, K_m values could not be obtained for low efficiency IDH1 enzymes because only saturating k_obs rates could be detected. In this case, k_obs rates are reported, which approximate k_cat rates. Results from assays at 21 °C are shown in supplemental Fig. S4. A, WT IDH1. B, H133Q IDH1. D, R100Q IDH1. E, R132H IDH1. F, R132C IDH1. G, R132G IDH1.