Publications

• Chemical confirmation of a pentavalent phosphorane in complex with beta-phosphoglucomutase.

  Tremblay L.W., Zhang G., Dai J., Dunaway-Mariano D., Allen K.N.

  This communication reports the X-ray crystal structure of the alpha-d-galactose-1-phosphate complex with that of Lactococcus lactis beta-phosphoglucomutase (beta-PGM) crystallized in the presence of Mg2+ cofactor and the enzyme-to-phosphorus ratio determined by protein and phosphate analyses of th ... >> More

  This communication reports the X-ray crystal structure of the alpha-d-galactose-1-phosphate complex with that of Lactococcus lactis beta-phosphoglucomutase (beta-PGM) crystallized in the presence of Mg2+ cofactor and the enzyme-to-phosphorus ratio determined by protein and phosphate analyses of the crystalline complex. The 1:1 ratio determined for this complex was compared to the 1:2 ratio determined for the crystals of beta-PGM grown in the presence of substrate and Mg2+ cofactor. This result verifies the published structure assignment of this latter complex as the phosphorane adduct formed by covalent bonding between the active site Asp8 carboxylate to the C(1)phosphorus of the beta-glucose 1,6-bisphosphate ligand and rules out the proposal of a beta-PGM-glucose-6-phosphate-1-MgF3- complex. << Less

  J. Am. Chem. Soc. 127:5298-5299(2005) [PubMed] [EuropePMC]

• The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction.

  Lahiri S.D., Zhang G., Dunaway-Mariano D., Allen K.N.

  Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in t ... >> More

  Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate. << Less

  Science 299:2067-2071(2003) [PubMed] [EuropePMC]

• Conformational cycling in beta-phosphoglucomutase catalysis: reorientation of the beta-D-glucose 1,6-(Bis)phosphate intermediate.

  Dai J., Wang L., Allen K.N., Radstrom P., Dunaway-Mariano D.

  Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniq ... >> More

  Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/-9 s(-)(1), K(m) = 49 +/- 4 microM for the substrate betaG1P and K(m) = 6.5 +/-0.7 microM for the activator beta-d-glucose 1,6-bisphosphate (betaG1,6bisP). The observed transient accumulation of [(14)C]betaG1,6bisP (12% at approximately 0.1 s) in the single turnover reaction carried out with excess betaPGM (40 microM) and limiting [(14)C]betaG1P (5 microM) and betaG1,6bisP (5 microM) supported the role of betaG1,6bisP as a reaction intermediate in the conversion of the betaG1P to G6P. Single turnover reactions of [(14)C]betaG1,6bisP with excess betaPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the betaG1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with betaPGM, [(14)C]betaG1P, and unlabeled betaG1,6bisP demonstrated complete exchange of label to the betaG1,6bisP during the catalytic cycle. Thus, the reorientation of the betaG1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of betaG1P suggest that the reorientation and phosphoryl transfer from betaG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of betaG1,6bisP to betaG1P plus G6P was examined to evidence a significant stabilization of betaPGM aspartyl phosphate. << Less

  Biochemistry 45:7818-7824(2006) [PubMed] [EuropePMC]

• Phosphoryl group transfer: evolution of a catalytic scaffold.

  Allen K.N., Dunaway-Mariano D.

  It is proposed that enzymic phosphoryl-transfer reactions occur by concerted, step-wise, associative (phosphorane-intermediate) or dissociative (metaphosphate-intermediate) mechanisms, as dictated by the catalytic scaffold and the reactants. During the evolution of a phosphotransferase family, the ... >> More

  It is proposed that enzymic phosphoryl-transfer reactions occur by concerted, step-wise, associative (phosphorane-intermediate) or dissociative (metaphosphate-intermediate) mechanisms, as dictated by the catalytic scaffold and the reactants. During the evolution of a phosphotransferase family, the mechanism of the phosphoryl-transfer reaction is in constant flux, potentially changing with each adaptation of the catalytic scaffold to a new phosphoryl-donor-acceptor pair. Phosphotransferases of the recently discovered haloacid dehalogenase superfamily of enzymes, one of the largest and most ubiquitous of the phosphotransferase families characterized to date, are described in the context of the co-evolution of the catalytic scaffold and mechanism. << Less

  Trends Biochem Sci 29:495-503(2004) [PubMed] [EuropePMC]

• Catalytic cycling in beta-phosphoglucomutase: a kinetic and structural analysis.

  Zhang G., Dai J., Wang L., Dunaway-Mariano D., Tremblay L.W., Allen K.N.

  Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the ... >> More

  Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/-20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell. << Less

  Biochemistry 44:9404-9416(2005) [PubMed] [EuropePMC]