Caught in the Act:  The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

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

doi:10.1021/bi0202373

Cited by 121 publications

(163 citation statements)

References 40 publications

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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: 71%

“…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: 71%

mentioning

confidence: 99%

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

et al. 2006

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“…The crystals were twinned and diffracted only to 8.0 Å on a RU300 X-ray generator. Subsequently, the conditions were optimized to 0.1 M Tris HCl, pH 7.0 and 1.5 M ammonium sulfate (at 18°C ) plus 100 mM vinyl sulfonate (vso 3 , an inhibitor) to produce larger, single crystals that grew within a week.…”

Section: Kinetic Characterization Of Phosphonatase and -Pgmmentioning

confidence: 99%

“…The two strong electron densities in the active site were assigned to vso 3 and Mg 2+ ions, respectively. The final R free and R work for the model were 26.0 and 23.9%, respectively.…”

Section: Kinetic Characterization Of Phosphonatase and -Pgmmentioning

confidence: 99%

“…These are L-2-haloacid dehalogenase (4), phosphonatase (1), -phosphoglucomutase ( -PGM) (3), the N-terminal domain (a lipid phosphatase) of the bifunctional mammalian phosphatase-epoxide hydrolase (5), the P-domain of the calcium pump ATPase (6), the phosphoserine phosphatase (7), the 3-deoxy-D-manno-octulsonate 8-phosphate phosphatase (8), the deoxyribonucleotidase and the C-terminal phosphatase domain of the bifunctional T4 polynucleotide kinase (9). Each of these enzymes contains a highly conserved R/ core domain, in which the active site is formed by four loops (Figure 1).…”

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Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain^,

et al. 2004

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The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the R/ core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight -turn in the helix-loophelix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and -phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.

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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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Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

Cited by 121 publications

References 40 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

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain^,

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

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Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

Cited by 121 publications

References 40 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

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain,

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

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Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain^,