E. coli MEP Synthase:  Steady-State Kinetic Analysis and Substrate Binding

• Published in: Biochemistry (Easton) Vol. 41; no. 1; pp. 236 - 243
• Main Authors: Koppisch, Andrew T, Fox, David T, Blagg, Brian S. J, Poulter, C. D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08-01-2002
• Subjects: Binding, Competitive; DNA Primers - chemistry; Enzyme Inhibitors; Erythritol - analogs & derivatives; Erythritol - metabolism; Escherichia coli; 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
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0118207

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 μM, and K M NADPH = 0.5 ± 0.2 μM. The rearrangement/reduction is reversible; K eq = 45 ± 6 for DXP and MEP at 150 μM 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.