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:  kcat = 1.9 ± 0.1 s-1, KmGAP = 0.068 ± 0.001 mM, and Kmpyruvate = 0.44 ± 0.05 mM for pyruvate and GAP; kcat = 1.7 ± 0.1 s-1, Kmd-glyceraldehyde = 33 ± 3 mM, and Kmpyruvate = 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...