title: Phosphoenolpyruvate mutase catalysis of phosphoryl transfer in phosphoenolpyruvate: kinetics and mechanism of phosphorus-carbon bond formation.
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 degrees C in the presence of Mg(II) are kcat = 5 s(-1), Km = 0.77 +/- 0.05 mM, and Keq = (2-9) x 10(-4). In the PEP forming direction, kcat = 100 s(-1) and Km = 3.5 +/- 0.1 microM. 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 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 (Ki = 180 +/- 10 microM) and oxalate (Ki = 32 +/- 1O microM) 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
authors.forename: J
authors.initials: J
authors.lastname: Kim
authors.forename: D
authors.initials: D
authors.lastname: Dunaway-Mariano
mesh_terms: D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D004734:Energy Metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008291:Malate Dehydrogenase / Q000378:metabolism; D015394:Molecular Structure; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism*; D010758:Phosphorus / Q000737:chemistry; D017875:Phosphotransferases (Phosphomutases) / Q000378:metabolism*; D013053:Spectrophotometry; D013769:Tetrahymena pyriformis / Q000201:enzymology; D013816:Thermodynamics
chemical_list: D010758:Phosphorus; D010728:Phosphoenolpyruvate; D002244:Carbon; D008291:Malate Dehydrogenase; D001219:Aspartate Aminotransferases; D017875:Phosphotransferases (Phosphomutases); C057167:phosphoenolpyruvate mutase