The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer Reaction

Lahiri, S. C.; Zhang, Guofeng; Dunaway‐Mariano, Debra; Allen, Karen N.

doi:10.1126/science.1082710

Cited by 315 publications

(289 citation statements)

References 42 publications

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“…Thus, by analogy, the aspartate would be in the correct position relative to the phosphorylated substrate for in-line nucleophilic attack at the phosphorus atom. This arrangement of ligands to WO 4 2-closely mirrors that found in the structures of WO 4 2--bound phosphonatase (PDB entry 1FEZ) (6), phosphate-bound phosphoserine phosphatase (PDB entry 1F5S) (7), glucose 1,6-(bis)phosphate-bound -PGM (PDB entry 1O03) (30), and phosphate-bound human mitochondrial deoxyribonucleotidase (PDB entry 1MH9) (31). The loop I amide nitrogen interactions are all retained as well as the loop II hydroxyl group of serine or threonine.…”

Section: Mg(ii) and Phosphate-binding Residues Of The Active Sitesupporting

confidence: 64%

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

et al. 2004

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The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 Å X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.

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Section: Mg(ii) and Phosphate-binding Residues Of The Active Sitesupporting

confidence: 64%

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

et al. 2004

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“…20 Stable five-coordinate oxovanadium species are a suitable mimic for the five-coordinate phosphoryl transition state proposed for a number of phosphorolytic reactions. 30,31 Previously, it was reported that the inhibition constant of pig PAP for vanadate is 40 μmol L -1 at pH 5.5, and the mode of binding of the inhibitor is noncompetitive. 23 The di-zinc alkaline phosphatase from Escherichia coli is inhibited by vanadate with similar efficiency (K i = 12 μmol L -1 at pH 8.0), and the crystal structure of the enzyme-inhibitor complex shows that vanadate binds in a penta-coordinate geometry to the active site with three of its oxygen atoms coordinating the two metal ions in the active site in a tripodal arrangement (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Elliott¹,

Mitić²,

Gahan³

et al. 2006

J. Braz. Chem. Soc.

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Fosfatases ácidas púrpuras (PAP) pertencem à família das metalo-hidrolases binucleares que catalisam a hidrólise de um grande grupo de substratos fosfoésteres em pH ácido. Apesar dos seus sítios ativos serem estruturamente conservados as PAP apresentam versatilidade mecanística. Neste trabalho são investigados alguns aspectos do mecanismo catalítico de duas PAP, usando os inibidores vanadato e fluoreto como sondas. Enquanto as magnitudes das constantes de inibição das duas enzimas pelo vanadato são semelhantes, as enzimas diferem com relação ao modo de inibição; vanadato interage de forma não-competitiva com a PAP de porco (K i = 40 µmol L -1 ), porém inibe a PAP de feijão competitivamente (K i = 30 µmol L -1 ). De modo semelhante, o fluoreto também age como inibidor competitivo da PAP de feijão, independentemente do pH, enquanto que o fluoreto simplesmente interage com o complexo formado pela enzima de porco e seu substrato(PAPsubstrato) em pH baixo e inibe de forma não competitiva esta enzima em pH mais alto, independentemente da composição do íon metálico. Além disso, enquanto a inibição pelo fluoreto se dá através da interação lenta com a PAP de porco, ele se liga rapidamente ao sítio catalítico da enzima do feijão. Visto que se propõe que o vanadato e o fluoreto mimetizem o estado de transição e o nucleófilo, respectivamente, as diferenças observadas na cinética de inibição indicam sutis, porém distintas diferenças no mecanismo de reação destas enzimas.Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (K i = 40 μmol L -1 ) while it inhibits red kidney bean PAP competitively (K i = 30 μmol L -1 ). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.Keywords: purple acid phosphatase, catalytic mechanism, kinetics, enzyme inhibition IntroductionPurple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases, with members differing widely with respect to physicochemical properties, tissue localisation...

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“…The rate and equilibrium for phosphorylation of the active site Asp8 by G16bisP are examined. A model for PGM catalysis is proposed that is based on cycling of the enzyme between the open and closed conformations observed in the reported PGM X-ray crystal structures (6,(12)(13)(14). lactis -phosphoglucomutase ( PGM) was prepared according to a published procedure (16).…”

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Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

et al. 2006

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Activated Lactococcus lactis -phosphoglucomutase ( PGM) catalyzes the conversion of -Dglucose 1-phosphate ( G1P) derived from maltose to -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 µM for the substrate G1P and K m ) 6.5 ( 0.7 µM for the activator -D-glucose 1,6-bisphosphate ( G1,6bisP). The observed transient accumulation of [ 14 C] G1,6bisP (12% at ∼0.1 s) in the single turnover reaction carried out with excess PGM (40 µM) and limiting [ 14 C] G1P (5 µM) and G1,6bisP (5 µM) supported the role of G1,6bisP as a reaction intermediate in the conversion of the G1P to G6P. Single turnover reactions of [ 14 C] G1,-6bisP with excess PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the G1,6bisP 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 PGM, [ 14 C] G1P, and unlabeled G1,6bisP demonstrated complete exchange of label to the G1,6bisP during the catalytic cycle. Thus, the reorientation of the G1,6bisP 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 G1P suggest that the reorientation and phosphoryl transfer from G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of G1,6bisP to G1P plus G6P was examined to evidence a significant stabilization of PGM aspartyl phosphate.Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate (G1P) 1 and D-glucose 6-phosphate (G6P). Operating in the forward G6P-forming direction, this reaction links polysaccharide phosphorolysis to glycolysis. In the reverse direction, the reaction provides G1P for the biosynthesis of exo-polysaccharides (2). There are two classes of phosphoglucomutases, the R-phosphoglucomutases (RPGM, EC 5.4.2.2), ubiquitous among eucaryotes and procaryotes, and the -phosphoglucomutases ( PGM, EC 5.4.2.6), present in certain bacteria and protists. The two classes of mutases are distinguished by their specificity for R-and -D-glucose phosphates and by their protein-fold family. The rabbit muscle RPGM (3) and the closely related Pseudomonas aeruginosa RPGM/RPMM (4) are members of the phosphohexomutase enzyme superfamily (5), whereas PGM (6) belongs to the haloalkanoic acid (HAD) enzyme superfamily (7). The four-domain RPGM and RPGM/RPMM (∼50 kDa) are approximately twice the size of the twodomain PGM (∼25 kDa).In RPGM (and in RPGM/RPMM), phosphoryl transfer is mediated by an active site serine which forms a stable phosphate ester...

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The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer Reaction

Cited by 315 publications

References 42 publications

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

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The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer Reaction

Cited by 315 publications

References 42 publications

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily,

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily,

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

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X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,

X-ray Crystal Structure of the Hypothetical Phosphotyrosine Phosphatase MDP-1 of the Haloacid Dehalogenase Superfamily^,