Kinetic Analysis of β-Phosphoglucomutase and Its Inhibition by Magnesium Fluoride

Goličnik, Marko; Olguín, Luis F.; Baxter, Nicola J.; Waltho, Jonathan P.; Williams, Nicholas H.; Hollfelder, Florian

doi:10.1021/ja806421f

Cited by 35 publications

(68 citation statements)

References 77 publications

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“…The 31 P NMR spectrum of β-PGM expressed and purified according to established procedures (13,24) showed that freshly prepared protein has no phosphate moiety. The addition of either G6P or βG1P to a solution of unliganded β-PGM, replicating the original crystallization conditions (17) (except that fluoride was omitted), failed to show any phosphorus species covalently bound to protein, as predicted by the detailed kinetic analysis of β-PGM (15). Under these conditions, there was also no measureable population of noncovalently bound sugar phosphate (only free G6P as α-and β-anomers, free Pi derived from the slow hydrolysis of G6P (15), and several other minor non-protein-bound sugar phosphates were observed).…”

Section: Resultsmentioning

confidence: 87%

“…The bacterial β-phosphoglucomutases (β-PGM, EC 5.4.2.6) are smaller proteins, operate on βG1P, and use a conserved aspartate (D8 in β-PGM from L. lactis) as a nucleophile to form a transient phospho-enzyme (13). The short lifetime of the phospho-enzyme (k hydrolysis ¼ 0.05 s −1 (14); 0.026 AE 0.001 s −1 (15)) is unsurprising because acyl phosphates are highly reactive phosphorylating species (ΔG ∘ hydrolysis ¼ −10 kcal mol −1 ). Only in exceptional circumstances are the half-lives extended above 24 h (16).…”

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confidence: 99%

“…mechanical -molecular mechanical (QM-MM) analysis based on the structure (19) and was subsequently validated in solution by direct observation based on 19 F NMR data (8). However, the MgF − 3 interpretation was questioned (20,21) and the pentacovalent phosphorane interpretation defended (22,23), despite the presence of fluoride, which severely compromises the catalytic cycle of β-PGM (15). Therefore, using an integration of NMR and high-resolution x-ray structural data, we now report a thorough characterization of β-PGM in the presence of G6P and fluoride that leaves no room for a pentacovalent phosphorane interpretation of solid-state or solution-state complexes.…”

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confidence: 99%

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Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

Baxter

Bowler

Alizadeh

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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137

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Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO − 3 , but is MgF − 3 (trifluoromagnesate). Although MgF − 3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19 F NMR. Measurements on three β-PGM-MgF − 3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F⋯H-N scalar couplings, and the atomic positions determined from the highresolution crystal structure of the β-PGM-MgF − 3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF − 3 model of near-transition state conformations.19F NMR | phosphoryl transfer enzyme | transition state analogue | trifluoromagnesate T he mono-and diesters of phosphoric acid have commanding and ubiquitous roles in all species of life. As structural components they show remarkable stability to spontaneous hydrolysis under near physiological conditions (25°C), with half-lives for P-O bond cleavage in phosphate diesters estimated at ca. 10 7 years and for monoesters ca. 10 12 years (1, 2). Yet, they are susceptible to enzyme-catalyzed hydrolysis and phosphoryl group transfer reactions either between two oxygens, or between oxygen and nitrogen or sulfur, with turnover numbers adequate to support a vast array of biological processes, e.g. Serratia nuclease k cat ca. 2; 500 s −1 (3), E. coli alkaline phosphatase k cat ≥ 45 s −1 (4), and human protein tyrosine phosphatase β k cat ca.

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Section: Resultsmentioning

confidence: 87%

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confidence: 99%

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confidence: 99%

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Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

Baxter

Bowler

Alizadeh

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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137

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“…This was then later examined by detailed NMR studies that also provided insight in the molecular mechanism of fluoride inhibition by bPGM (Baxter et al 2006), as well as further (steady and pre-steady state) kinetic analysis of both the reactivity of bPGM and its inhibition by magnesium fluoride, which essentially ruled out the existence of a stable pentavalent phosphorane intermediate in the active site of bPGM (Golicnick et al 2009). Recent theoretical studies would also seem to support this finding (Marcos et al 2010).…”

Section: B-phosphoglucomutase (Bpgm) : Metal Fluoride Ts Analogs As Pmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

339

448

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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“…Phosphoisomerism is achieved through the release of either glucose 6-phosphate (G6P) or β-glucose 1-phosphate (βG1P) from the resulting phosphoenzyme complex, according to Scheme 1. Work to date has focused on probing the reaction mechanism of β-PGM through the introduction of metal fluoride transition-state analogs (TSAs) and computational analysis based on the derived structural data (15)(16)(17)(18)(19)(20)(21). In particular, trifluoromagnesate (MgF 3 − ) has been established as a very close steric and electronic mimic of the planar PO 3 − group (16,20,(22)(23)(24) that represents the energy maximum as phosphorus moves through the plane of the three PO 3 − oxygen atoms (15,21).…”

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confidence: 99%

Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate

Griffin

Bowler

Baxter

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Experimental observations of fluoromagnesate and fluoroaluminate complexes of β-phosphoglucomutase (β-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of β-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.ground-state analogues | enzyme mechanism T he stabilization of transition states (TS) is central to the high reaction rates achieved by enzymes (1-3). Most studies to date have focused on TSs that include a chemical transformation step, where the reacting species have some partial chemical bonding character. However, enzymes also have to avoid substantial barriers associated with nonchemical transformation steps and hence may be required to stabilize other regions of the complex energy surface corresponding to the reaction coordinate. The concept of near attack conformers (NACs) was introduced as a useful tool to help analyze these different roles, by partitioning chemical transformation and nonchemical transformation steps (4). NACs were defined as species where the reactants have an appropriate geometry for the development of the chemical TS (5, 6). Furthermore, the component of the TS barrier ascribed to the chemical TS was proposed to be relatively constant within systems carrying out similar chemistry, on the basis of a linear relationship between the (calculated) population of NACs and the corresponding reaction rates (5). Hence, in free-energy terms, the NAC model apportions the TS barrier (ΔG † ) between a variable conformational component (ΔG NAC ) and an invariant chemical (covalent) component (ΔG chem ; Fig. 1). For some uncatalyzed reactions, it was proposed that a major component of the TS free-energy barrier is the population of NACs, t...

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Kinetic Analysis of β-Phosphoglucomutase and Its Inhibition by Magnesium Fluoride

Cited by 35 publications

References 77 publications

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

Why nature really chose phosphate

Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate

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