Evidence for an Intramolecular, Stepwise Reaction Pathway for PEP Phosphomutase Catalyzed P-C Bond Formation

Article abstract of DOI:10.1021/jo00025a031

The Tetrahymena pyriformis enzyme, phosphoenolpyruvate phosphomutase, catalyzes the rearrangement of phosphoenolpyruvate to the P-C bond containing metabolite, phosphonopyruvate.To distinguish between an intra- and intermolecular reaction pathway for this process an equimolar mixture of 18O,C(2)-¹⁸O>thiophosphonopyruvate and (all ¹⁶O) thiophosphonopyruvate was reacted with the phosphonomutase, and the resulting products were analyzed by ³¹P NMR.The absence of the cross-over product 18O>thiophosphonoenolpyruvate in the product mixture was interpreted as evidence for an intramolecular reaction pathway.To distinguish between a concerted and stepwise intramolecular reaction pathway the pure enantiomers of the chiral substrate <¹⁸O>-thiophosphonopyruvate were prepared and the stereochemicalb course of their conversion to chiral <¹⁸O>thiophosphoenolpyruvate was determined.The assignments of the phosphorous configurations in the <¹⁸O>thiophosphonopyruvate enantiomers reported earlier (McQueney, M.S.; Lee, S.-l.; Bowman, E.; Mariano, P.S.; Dunaway- Mariano, D.J.Am.Chem.Soc. 1989, 111, 6885-6887) were revised according to the finding that introduction of the ¹⁸O label into the thiophosphonopyruvate precursor occurs with retention rather than with (the previously assumed) inversion of configuration.On the basis the observed conversion of (S_p)-<¹⁸O>thiophosphonopyruvate to (S_p)-<¹⁸O>thiophosphonoenolpyruvate and (R_p)-<¹⁸O>thiophosphonopyruvate to (R_p)-<¹⁸O>thiophosphonoenolpyruvate, it was concluded that the PEP phosphomutase reaction proceeds with retention of the phosphorus configuration and therefore by a stepwise mechanism.Lastly, the similar reactivity of the oxo- and thio-substituted phosphonopyruvate substrates (i.e., nearly equal V_max) was interpreted to suggest that nucleophilic addition to the phosphorus atom is not rate limiting among the reaction steps.