Rhodobacter capsulatus 1-Deoxy-d-xylulose 5-Phosphate Synthase:  Steady-State Kinetics and Substrate Binding

1-Deoxy-d-xylulose 5-phosphate synthase (DXP synthase) catalyzes the thiamine diphosphate (TPP)-dependent condensation of pyruvate and d-glyceraldehyde 3-phosphate (GAP) to yield DXP in the first step of the methylerythritol phosphate pathway for isoprenoid biosynthesis. Steady-state kinetic constants for DXP synthase calculated from the initial velocities measured at varying concentrations of substrates were as follows: k cat = 1.9 ± 0.1 s-1, K m GAP = 0.068 ± 0.001 mM, and K m pyruvate = 0.44 ± 0.05 mM for pyruvate and GAP; k cat = 1.7 ± 0.1 s-1, K m d-glyceraldehyde = 33 ± 3 mM, and K m pyruvate = 1.9 ± 0.5 mM for d-glyceraldehyde and pyruvate. β-Fluoropyruvate was investigated as a dead-end inhibitor for pyruvate. Double-reciprocal plots showed a competitive inhibition pattern with respect to pyruvate and noncompetitive inhibition with respect to GAP/d-glyceraldehyde. 14CO2 trapping experiments demonstrated that the binding of both substrates (pyruvate and GAP/d-glyceraldehyde) is required for the formation of a catalytically competent enzyme−substrate complex. These results are consistent with an ordered mechanism for DXP synthase where pyruvate binds before GAP/d-glyceraldehyde.