Phosphoenolpyruvate mutase catalysis of phosphoryl transfer in phosphoenolpyruvate: Kinetics and mechanism of phosphorus-carbon bond formation

Phosphoenolpyruvate phosphomutase (PEP mutase) from Tetrahymena pyriformis catalyzes the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr). A spectrophotometric P-pyr assay consisting of the coupled actions of P-pyr decarboxylase, phosphonoacetaldehyde hydrolase, and alcohol dehydrogenase was devised to monitor mutase catalysis. The reaction constants determined for PEP mutase catalyzed conversion of PEP to P-pyr at pH 7.5 and 25 degrees C in the presence of MS(II) are k(cat) = 5 s(-1), K-m = 0.77 +/- 0.05 mM, and K-eq = (2-9) x 10(-4) In the PEP forming direction, k(cat) = 100 (-1) and K-m = 3.5 +/- 0.1 mu M. Retention of stereochemistry at phosphorus and strong inhibition displayed s by the pyruvyl enolate analog, oxalate, have been cited as two lines of evidence that PEP mutase catalysis proceeds via a phosphoenzyme-pyruvyl enolate intermediate [Seidel, H. M., & Knowles, J. R. (1994) Biochemistry 33, 5641-5646]. In this study, single turnover reactions of oxalyl phosphate with the PEP mutase were carried out to test the formation of the phosphoenzyme intermediate. If formed, the phosphoenzyme-oxalate complex should be sufficiently stable to isolate. Reaction of the mutase with [P-32]oxalyl phosphate in the presence of Mg(II)/Mn(II) cofactor failed to produce a detectable level of the [P-32]phosphoenzyme-oxalate complex. In contrast, the same reaction carried out with pyruvate phosphate dikinase (PPDK), an enzyme known to catalyze the phosphorylation of its active site histidine with PEP, occurred at a rate of 4 x 10(-4) s(-1) (15% E-P formed) in the presence Mg(II) and at a rate of 3 x 10(-3) s(-1) (60% E-P formed) in the presence of Mn(II). Both oxalyl phosphate (K-i = 180 +/- 10 mu M) and oxalate (K-i = 32 +/- 10 mu M) were competitive inhibitors of PEP mutase catalysis, but neither displayed slow, tight binding inhibition. These results do not support the intermediacy of a phosphoenzyme-pyruvyl enolate complex in PEP mutase catalysis.