title: Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli.
abstract: The mechanism of the reaction catalyzed by 2-C-methyl-d-erythritol 4-phosphate (MEP) synthase from Escherichia coli has been studied by steady-state and single-turnover kinetic experiments for the 1-deoxy-d-xylulose 5-phosphoric acid (DXP) analogues, 1,1,1-trifluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(3)-DXP), 1,1-difluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(2)-DXP), 1-fluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-d-hexulose 6-phosphate (Et-DXP). CF(3)-DXP, CF(2)-DXP, and Et-DXP were poor inhibitors, most likely because of the increase in steric bulk at C1 of DXP. The three analogues were also poor substrates for the enzyme. In contrast, CF-DXP was a good substrate (k(cat)(CF)(-)(DXP) = 37 +/- 2 s(-)(1), K(m)(CF)(-)(DXP) = 227 +/- 25 microM) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-)(1), K(m)(DXP) = 45 +/- 4 microM). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[(2)H]NADPH ((H)k/(D)k = 1.34 +/-0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[(2)H]NADPH ((H)k/(D)k = 1.02 +/- 0.02). The reaction did not exhibit burst kinetics for either substrate, indicating that product release is not rate-limiting. These studies suggest that positive charge does not develop at C2 of DXP during catalysis. In addition, the isotope effect with CF-DXP and 4S-[(2)H]NADPH but not DXP indicates that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially rate-limiting for CF-DXP. Thus, rearrangement appears to be enhanced by substitution of a hydrogen atom in the methyl group of DXP by fluorine. These observations are consistent with a retro-aldol/aldol mechanism for the rearrangement during conversion of DXP to MEP.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Fox
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000737:chemistry; D019747:Aldose-Ketose Isomerases / Q000737:chemistry*; D002384:Catalysis; D041961:Deuterium Exchange Measurement; D004896:Erythritol / Q000031:analogs & derivatives / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005459:Fluorides / Q000737:chemistry; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry*; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D010428:Pentosephosphates / Q000737:chemistry; D010756:Phosphoric Acids / Q000737:chemistry; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000737:chemistry
chemical_list: C109460:1-deoxylulose 5-phosphate; C114232:2-C-methylerythritol 4-phosphate; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010428:Pentosephosphates; D010756:Phosphoric Acids; D013403:Sugar Phosphates; C081381:difluorophosphoric acid; D009249:NADP; C001308:glycolaldehyde phosphate; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D005634:Fructose-Bisphosphate Aldolase; D019747:Aldose-Ketose Isomerases; D000079:Acetaldehyde; D005459:Fluorides; D004896:Erythritol