E. coli MEP synthase: steady-state kinetic analysis and substrate binding.
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.
Koppisch
Andrew T
AT
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Fox
David T
DT
Blagg
Brian S J
BS
Poulter
C D
CD
D001667:Binding, Competitive; D017931:DNA Primers / Q000737:chemistry; D004791:Enzyme Inhibitors; D004896:Erythritol / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005578:Fosfomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010428:Pentosephosphates / Q000378:metabolism; D010640:Phenothiazines; D011485:Protein Binding; D011994:Recombinant Proteins; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C109460:1-deoxylulose 5-phosphate; C114232:2-C-methylerythritol 4-phosphate; D017931:DNA Primers; D004791:Enzyme Inhibitors; D010428:Pentosephosphates; D010640:Phenothiazines; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D005578:Fosfomycin; D009249:NADP; C024640:fosmidomycin; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D004896:Erythritol; C006069:mequitazine