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. beta-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. (14)CO(2) 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.
Eubanks
Lisa M
LM
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Poulter
C Dale
CD
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002245:Carbon Dioxide / Q000737:chemistry; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry; D005985:Glyceraldehyde / Q000737:chemistry; D005986:Glyceraldehyde 3-Phosphate / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D011773:Pyruvates / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013379:Substrate Specificity; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011773:Pyruvates; D011994:Recombinant Proteins; C017319:3-fluoropyruvate; D005986:Glyceraldehyde 3-Phosphate; D002245:Carbon Dioxide; D005985:Glyceraldehyde; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase