PMID:11772021
| Citation |
Koppisch, AT, Fox, DT, Blagg, BS and Poulter, CD (2002) E. coli MEP synthase: steady-state kinetic analysis and substrate binding. Biochemistry 41:236-43 |
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| Abstract |
2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM. The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP. |
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| Keywords |
Binding, Competitive; DNA Primers/chemistry; Enzyme Inhibitors; Erythritol/analogs & derivatives; Erythritol/metabolism; Escherichia coli/enzymology; Fosfomycin/analogs & derivatives; Fosfomycin/pharmacology; Kinetics; NADP/metabolism; Oxidation-Reduction; Pentosephosphates/metabolism; Phenothiazines; Protein Binding; Recombinant Proteins; Substrate Specificity; Sugar Phosphates/metabolism; Transferases/genetics; Transferases/isolation & purification; Transferases/metabolism |
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