Publications

• Purification and characterization of the Tetrahymena pyriformis P-C bond forming enzyme phosphoenolpyruvate phosphomutase.

  Bowman E.D., McQueney M.S., Scholten J.D., Dunaway-Mariano D.

  In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoenolpyruvate phosphomutase are described. PEP phosphomutase was first fractionated from T. pyriformis cellular extract by using 70% ammonium sulfate. Chromatography of the crude protein fraction on a DE ... >> More

  In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoenolpyruvate phosphomutase are described. PEP phosphomutase was first fractionated from T. pyriformis cellular extract by using 70% ammonium sulfate. Chromatography of the crude protein fraction on a DEAE-cellulose column followed by phenyl-Sepharose column chromatography and then Bio-Gel P-200 column chromatography afforded pure PEP phosphomutase in an approximate overall yield of 70 units/150 g of cells. The maximum turnover number observed for PEP phosphomutase catalysis of the phosphonopyruvate----PEP reaction is 38 s-1 (25 degrees C). The enzyme was shown to be a homodimer of 38,000-dalton subunits and to require a divalent metal ion for activity. Mg2+ (relative Vm = 1), Co2+ (rel Vm = 0.5), Zn2+ (rel Vm = 0.4), and Mn2+ (rel Vm = 0.3) each satisfied the cofactor requirement. Binding of the physiological cofactor, Mg2+ (Ki = 0.3 mM at pH 7.5), and phosphonopyruvate (Km = 2 microM at pH 7.5) was found to be ordered, with cofactor binding preceding substrate binding. Within the pH range of 5-9 catalysis (Vm) was found to be pH independent, while phosphonopyruvate binding dropped at acidic and basic pH. << Less

  Biochemistry 29:7059-7063(1990) [PubMed] [EuropePMC]

• Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

  Jia Y., Lu Z., Huang K., Herzberg O., Dunaway-Mariano D.

  PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus eduli ... >> More

  PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. << Less

  Biochemistry 38:14165-14173(1999) [PubMed] [EuropePMC]

• Helix swapping between two alpha/beta barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg(2+)-oxalate.

  Huang K., Li Z., Jia Y., Dunaway-Mariano D., Herzberg O.

  <h4>Background</h4>Phosphonate compounds are important secondary metabolites in nature and, when linked to macromolecules in eukaryotes, they might play a role in cell signaling. The first obligatory step in the biosynthesis of phosphonates is the formation of a carbon-phosphorus bond by convertin ... >> More

  <h4>Background</h4>Phosphonate compounds are important secondary metabolites in nature and, when linked to macromolecules in eukaryotes, they might play a role in cell signaling. The first obligatory step in the biosynthesis of phosphonates is the formation of a carbon-phosphorus bond by converting phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr), a reaction that is catalyzed by PEP mutase. The PEP mutase functions as a tetramer and requires magnesium ions (Mg2+).<h4>Results</h4>The crystal structure of PEP mutase from the mollusk Mytilus edulis, bound to the inhibitor Mg(2+)-oxalate, has been determined using multiwavelength anomalous diffraction, exploiting the selenium absorption edge of a selenomethionine-containing protein. The structure has been refined at 1.8 A resolution. PEP mutase adopts a modified alpha/beta barrel fold, in which the eighth alpha helix projects away from the alpha/beta barrel instead of packing against the beta sheet. A tightly associated dimer is formed, such that the two eighth helices are swapped, each packing against the beta sheet of the neighboring molecule. A dimer of dimers further associates into a tetramer. Mg(2+)-oxalate is buried close to the center of the barrel, at the C-terminal ends of the beta strands.<h4>Conclusions</h4>The tetramer observed in the crystal is likely to be physiologically relevant. Because the Mg(2+)-oxalate is inaccessible to solvent, substrate binding and dissociation might be accompanied by conformational changes. A mechanism involving a phosphoenzyme intermediate is proposed, with Asp58 acting as the nucleophilic entity that accepts and delivers the phosphoryl group. The active-site architecture and the chemistry performed by PEP mutase are different from other alpha/beta-barrel proteins that bind pyruvate or PEP, thus the enzyme might represent a new family of alpha/beta-barrel proteins. << Less

  Structure 7:539-548(1999) [PubMed] [EuropePMC]

• Dissociative phosphoryl transfer in PEP mutase catalysis: structure of the enzyme/sulfopyruvate complex and kinetic properties of mutants.

  Liu S., Lu Z., Jia Y., Dunaway-Mariano D., Herzberg O.

  The crystal structure of PEP mutase from Mytilus edulis in complex with a substrate-analogue inhibitor, sulfopyruvate S-pyr (K(i) = 22 microM), has been determined at 2.25 A resolution. Mg(II)-S-pyr binds in the alpha/beta barrel's central channel, at the C-termini of the beta-strands. The binding ... >> More

  The crystal structure of PEP mutase from Mytilus edulis in complex with a substrate-analogue inhibitor, sulfopyruvate S-pyr (K(i) = 22 microM), has been determined at 2.25 A resolution. Mg(II)-S-pyr binds in the alpha/beta barrel's central channel, at the C-termini of the beta-strands. The binding mode of S-pyr's pyruvyl moiety resembles the binding mode of oxalate seen earlier. The location of the sulfo group of S-pyr is postulated to mimic the phosphonyl group of the product phosphonopyruvate (P-pyr). This sulfo group interacts with the guanidinium group of Arg159, but it is not aligned for nucleopilic attack by neighboring basic amino side chains. Kinetic analysis of site directed mutants, probing the key active site residues Asp58, Arg159, Asn122, and His190 correlate well with the structural information. The results presented here rule out a phosphoryl transfer mechanism involving a double displacement, and suggest instead that PEP mutase catalysis proceeds via a dissociative mechanism in which the pyruvyl C(3) adds to the same face of the phosphorus from which the C(2)O departs. We propose that Arg159 and His190 serve to hold the phosphoryl/metaphosphate/phosphonyl group stationary along the reaction pathway, while the pyruvyl C(1)-C(2) bond rotates upon formation of the metaphosphate. In agreement with published data, the phosphoryl group transfer occurs on the Si-face of PEP with retention of configuration at phosphorus. << Less

  Biochemistry 41:10270-10276(2002) [PubMed] [EuropePMC]

• Interaction of inhibitors with phosphoenolpyruvate mutase: implications for the reaction mechanism and the nature of the active site.

  Seidel H.M., Knowles J.R.

  The active site and mechanism of action of the enzyme phosphoenolpyruvate mutase have been probed using substrate and intermediate analogues as inhibitors of the mutase-catalyzed reaction. Smaller anions (e.g. sulfite, nitrate, phosphinate, and bicarbonate) are noncompetitive inhibitors of the mut ... >> More

  The active site and mechanism of action of the enzyme phosphoenolpyruvate mutase have been probed using substrate and intermediate analogues as inhibitors of the mutase-catalyzed reaction. Smaller anions (e.g. sulfite, nitrate, phosphinate, and bicarbonate) are noncompetitive inhibitors of the mutase, while larger anions in the complementary series (sulfate, phosphonate, phosphate) inhibit competitively. Combining oxalate, an intermediate analogue that is a potent inhibitor of the mutase (Ki = 25 microM), with small, noncompetitive inhibitor anions results in synergistic inhibition of the mutase, suggesting that the combined presence of oxalate and anion creates a "bimolecular transition-state analogue". The phosphoenolpyruvate (PEP) mutase genes from Tetrahymena and Streptomyces are known, and these enzymes share significant amino acid sequence similarity to the isocitrate lyase gene from Ricinus. Despite their seeming structural unrelatedness to the substrates of PEP mutase, several isocitrate analogues are good inhibitors, suggesting that isocitrate lyase and PEP mutase are evolutionarily related. An active-site model has been developed that is in accord with the data presented, which are consistent with a mechanism involving the intermediacy of a phosphoenzyme. << Less

  Biochemistry 33:5641-5646(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.