Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli

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 (CF3-DXP), 1,1-difluoro-l-deoxy-D-Xylulose 5-phosphoric acid (CF2-DXP). 1-fluoro1-deoxy-D-Xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-D-hexulose 6-phosphate (Et-DXP). CF3-DXP, CF2-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 mu M) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-1), K-m(DXP) = 45 +/- 4 mu M). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[H-2]NADPH ((11)k/Dk = 1.34 +/- 0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[H-2]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-[H-2]NADPH but not DXP indica(es that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially ratelimiting 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.