Stereochemical Analysis of the Tetrahedral Adduct Formed at the Active Site of UDP-GlcNAc Enolpyruvyl Transferase from the Pseudosubstrates, (E)- and (Z)-3-Fluorophosphoenolpyruvate, in D_2O

摘要

Of the remarkably diverse chemical transformations that phosphoenolpyruvate (PEP) undergoes in metabolism, the enzymic formation of an enol ether through the intact transfer of the enolpyruvyl group of PEP to a cosubstrate alcohol is notable in that there is no net electrophilic addition to C-3 of PEP. UDP-GlcNAc enolpyruvyl transferase (MurZ), which catalyzes the first committed step in bacterial peptidoglycan assembly, and 5-enolpyruvylshikimate-3-phosphate (EPSP) syn-thase of the shikimic acid pathway are the two enzymes known to catalyze this unusual enopyruvyl transfer and have been the subject of considerable mechanistic investigation, in part because each enzyme constitutes the site of action of important inhibitors: the antibiotic fosfomycin inactivates MurZ, and the herbicide glyphosate inhibits EPSP synthase. The transient and reversible formation of a methyl group by protonation of C-3 of PEP has been demonstrated by the isolation of a tetrahedral ketal intermediate in both the EPSP synthase and MurZ reaction pathways by rapid quench methods, establishing an addition-elimination mechanism for enolpyruvyl transfer (Scheme 1). The relative stereochemistry of the enzyme-catalyzed