Phosphoenolpyruvate Mutase Catalysis of Phosphoryl Transfer in Phosphoenolpyruvate: Kinetics and Mechanism of Phosphorus−Carbon Bond Formation

Publisher Website
Google Scholar
Abstract
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 °C in the presence of Mg(II) are kcat = 5 s-1, Km = 0.77 ± 0.05 mM, and Keq = (2−9) × 10-4. In the PEP forming direction, kcat = 100 s-1 and Km = 3.5 ± 0.1 μM. Retention of stereochemistry at phosphorus and strong inhibition displayed 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 [32P]oxalyl phosphate in the presence of Mg(II)/Mn(II) cofactor failed to produce a detectable level of the [32P]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 × 10-4 s-1 (15% E-P formed) in the presence Mg(II) and at a rate of 3 × 10-3 s-1 (60% E-P formed) in the presence of Mn(II). Both oxalyl phosphate (Ki = 180 ± 10 μM) and oxalate (Ki = 32 ± 10 μM) 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.

This publication has 32 references indexed in Scilit: