Catalytic Cycling in β-Phosphoglucomutase:  A Kinetic and Structural Analysis<sup>,</sup>

Dai, Jianying; Wang, Liangbing; Dunaway‐Mariano, Debra; Tremblay, L.W.; Allen, Karen N.

doi:10.1021/bi050558p

Cited by 53 publications

(139 citation statements)

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“…This is also the case for a partially open conformation that has been observed in the inactive complex formed between the unphosphorylated PGM and R-Dgalactose-1-phosphate (the C(6)OH is positioned near the Asp8 and the C(1)phosphate at the domain-domain interface) (14). X-ray structures of the apo-native enzyme (12) and the apo-phosphorylated enzyme (6) The reactions carried out under single turnover conditions employed enzyme concentrations that equaled or exceeded the reactant concentrations. Thus, the ratios of reactant; the product; and the G1,6bisP activator are determined not only by their intrinsic energies but also by the intrinsic energies of E and E-P, and by the binding energy associated with the various enzyme-ligand complexes.…”

Section: G16bisp Is An Intermediate In the Pgm-catalyzed Reactionmentioning

confidence: 72%

mentioning

confidence: 99%

“…2 This reaction is mediated by an active site aspartate (Asp8), which forms an acyl phosphate as the covalent enzyme intermediate (12). Kinetic methods were used to demonstrate the intermediacy of G16bisP in G6P formation and to show that the G16bisP intermediate is reoriented in the active site by dissociation into solvent and then binding in the opposite orientation.…”

mentioning

confidence: 99%

“…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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confidence: 99%

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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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Section: G16bisp Is An Intermediate In the Pgm-catalyzed Reactionmentioning

confidence: 72%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

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

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“…This approach allows us to distinguish MgF 3 Ϫ from the central PO 3 Ϫ moiety in INT unambiguously. When ␤-PGM is added to a solution containing MgCl 2 and a 10-fold excess of NH 4 exhibiting the largest chemical shift changes correspond to those residues which comprise the active site loops, or else residues located in the ''hinge'' region between the ''cap'' and ''core'' domains (10). These NMR observations are consistent with the suggestion that these domains move as rigid bodies relative to each other to enclose the substrate (10).…”

Section: Resultsmentioning

confidence: 99%

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

Baxter

Olguín

Goličnik

et al. 2006

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

113

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Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. ␤-Phosphoglucomutase catalyses the isomerization of ␤-glucose-1-phosphate to ␤-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19 F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF 3 ؊ mimics the transferring PO 3 ؊ moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of ␤-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.enzyme mechanism ͉ fluoride inhibition ͉ NMR structure ͉ phosphoryl transfer ͉ isosteric isoelectronic ͉ transition state analogue P hosphate transfer reactions play a central role in metabolism, regulation, energy housekeeping and signaling (1). As phosphate esters are kinetically extremely stable, efficient catalysis is crucial for the control of these cellular processes. Although model studies have taught us much about the intrinsic chemical mechanisms (2), our understanding of the origins of the enormous enzymatic rate accelerations involved, up to a factor of 10 21 (3), is far from complete (4). A snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed (5). However, is this realistic given how elusive high-energy intermediates and transition states (TSs) inevitably are? The direct observation of TSs for simple organic reactions has required ultrafast lasers with femtosecond resolution (6) and no physical or spectroscopic method is available to observe the structure of TSs of enzymatic reactions directly. Thus transition state analogues that bind tightly in an enzyme active site have been of paramount importance in defining the structural and energetic framework for catalysis (7,8).An observation that appears to challenge this paradigm arises from structural studies with ␤-phosphoglucomutase (␤-PGM, EC 5.4.2.6): namely, that a high-energy phosphorane on the reaction pathway has been observed directly by x-ray crystallography, demonstrating how the enzyme interacts with a very high-energy, metastable species (9). The latter also apparently demonstrated that the enzyme catalyzed reaction proceeds through an addition-elimination mechanism, a reaction pathway not observed in solution for phosphate monoester anions. However, the observation of an enzyme "caught in the act" is surprising: the demands of turnover mean that the enzyme would gain no apparent advant...

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Covalent Nucleophilic Catalysis: Structures of Enzyme Intermediates in Covalent Enzyme Catalysis

Ringe¹

2021

Encyclopedia of Life Sciences

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Enzyme catalysis relies on several factors, such as proper fit of the substrate to the enzyme active site and interactions between substrate and residues that will aid in the chemical reaction. The overall effect is to lower the energy barrier for the reaction. One factor that is observed in some reactions is the formation of an intermediate covalent adduct to a residue in the active site in what is considered the first half of the reaction. This intermediate then goes on to react with another substrate to finish the overall reaction. Sometimes these covalent intermediates can be captured and characterised structurally. The chemical structure of an intermediate can define how a reaction proceeds, how the enzyme responds to binding of substrate, which residues of the active site are involved in the chemical transformation and how the substrate undergoes the rearrangements needed to produce the products. Capture of intermediates is not straightforward and requires different strategies, such as trapping with altered reaction conditions, use of low temperature and the use of mutants that prevent the second half of the reaction to proceed. A number of residues are able to form adducts to a ligand, depending on the nucleophilicity of the atom which undergoes the addition to a substrate (or part of a substrate). Key Concepts Covalent intermediates can lower the energy barrier in an enzyme‐catalysed reaction. Catalysis by an enzyme allows a reaction to occur under conditions that would be unfavourable or exceedingly slow in the absence of the enzyme. Strategies to accomplish this feat depend on the structure and physical properties of the protein to promote precise orientation of a substrate relative to the catalytic residues in an active site and stabilising unstable species with polarising bonds and forming covalent adducts. Structures of covalent intermediates help to define the mechanism by which an enzyme achieves its catalytic power. The structure of covalent intermediates can be trapped by a number of strategies, such as altering reaction conditions such as pH, by drastically lowering the temperature in order to prevent the second half of the reaction to occur or using mutants that prevent the second half of the reaction to occur. The most common covalent intermediates involve acylation, phosphorylation, glycosylation and Schiff base formation. Formation of a covalent adduct can lower the energy barrier of a reaction.

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Catalytic Cycling in β-Phosphoglucomutase: A Kinetic and Structural Analysis^,

Cited by 53 publications

References 73 publications

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

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

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

Covalent Nucleophilic Catalysis: Structures of Enzyme Intermediates in Covalent Enzyme Catalysis

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Catalytic Cycling in β-Phosphoglucomutase: A Kinetic and Structural Analysis,

Cited by 53 publications

References 73 publications

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

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

A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active site

Covalent Nucleophilic Catalysis: Structures of Enzyme Intermediates in Covalent Enzyme Catalysis

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Catalytic Cycling in β-Phosphoglucomutase: A Kinetic and Structural Analysis^,

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site