The fate of the hydrogens of phosphoenolpyruvate in the reaction catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase: isotope effects and isotope exchange

Abstract The condensation reaction of phosphoenolpyruvate and shikimate 3-phosphate catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase is thought to proceed by an addition-elimination mechanism in which C-3 of phosphoenolpyruvate transiently becomes a methyl group in the enzyme-bound intermediate. Results obtained from reactions conducted in H2O, 2H2O, and 3H2O, using unlabeled, [3-2H2]-, or [3-3H,2H]phosphoenolpyruvate, are consistent with the addition-elimination pathway and show that the transient methyl group rotates rapidly. There is substantial discrimination against heavy hydrogen isotopes in both the protonation and deprotonation steps. These results demonstrate the feasibility of determining the stereochemical course of the synthase reaction.