E-coli MEP synthase:: Steady-state kinetic analysis and substrate binding

被引：116

作者：

Koppisch, AT ^{[1
]}

Fox, DT ^{[1
]}

Blagg, BSJ ^{[1
]}

Poulter, CD ^{[1
]}

机构：

[1] Univ Utah, Dept Chem, Salt Lake City, UT 84112 USA

来源：

BIOCHEMISTRY | 2002年 / 41卷 / 01期

关键词：

D O I：

10.1021/bi0118207

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/ reduction of I -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 muM, and K-M(NADPH) = 0.5 +/- 0.2 /M. The rearrangement/reduction is reversible; K-eq = 45 +/- 6 for DXP and MEP at 150 muM 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 it pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.

引用

页码：236 / 243

页数：8

共 52 条

[1] Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase:: an approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa [J].