Analysis of Fluoromethyl Group Chirality Establishes a Common Stereochemical Course for the Enolpyruvyl Transfers Catalyzed by EPSP Synthase and UDP-GlcNAc Enolpyruvyl Transferase

Abstract

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [¹H,²H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with our prior analogous work on the MurA reaction [Kim, D. H., Lees, W. J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380−6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.