Structural and mechanistic studies of enolase

Reed, George H.; Poyner, Russell R.; Larsen, Todd M.; Wedekind, Joseph E.; Rayment, Ivan

doi:10.1016/s0959-440x(96)80002-9

Cited by 88 publications

(95 citation statements)

References 26 publications

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“…The so-called conformational ions ensure the proper spatial structure of the active center, whereas the catalytic ions are necessary for the reaction mechanism [45,46]. They ensure the insertion of the substrate into catalytic pocket and its transformation to the reaction product [50]. Since methylglyoxal is, like the glycolytic substrate 2-PGA, a tricarbon molecule, it can get to the catalytic center.…”

Section: Discussionmentioning

confidence: 99%

“…The phosphate residue of 2-PGA is a very important functional group for the insertion and proper orientation of the substrate to the active center of the enzyme. The phosphate group interacts with the amino-acid residues of the catalytic pocket and conformational magnesium ions, which ensures the transformation of 2-PGA into an enol intermediate and the formation of the reaction product PEP [50].…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Gamian

Staniszewska

Danielewicz

2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolasederived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K M value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V max and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Gamian

Staniszewska

Danielewicz

2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…Lys345 (E. coli: Lys341) and Glu211 (E. coli: Glu208) have been proposed to act as a catalytic base-acid pair. 17,23 An alternative proposal suggests that Glu211 and Glu168 share a proton, which then catalyzes the abstraction of the hydroxy group. 18 In yeast and lobster enolase, it has been observed that the binding of substrate causes major conformational changes in the enzyme that closes the substrate into the active site.…”

Section: The Active Sitementioning

confidence: 99%

Crystal structure of the Escherichia coli RNA degradosome component enolase

Kühnel

Luisi

2001

Journal of Molecular Biology

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The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A Ê . There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules,¯exible loops close onto the active site. This closure mimics the conformation of the substratebound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a speci®c role to it, a structural role is possible.

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“…5 Previous experiments showed that K345A and K345M variants and the E211Q variant are inactive in the overall reaction. 3,6 A recent structure of E211Q showed proper folding, 5 but all previous attempts to obtain crystals of K345A (or other variants of this critical residue) have proven unsuccessful. Since enolase exists as a homodimer in most organisms, a new strategy involving engineered heterodimeric enolases was adopted.…”

Section: Introductionmentioning

confidence: 99%

Structure and Catalytic Properties of an Engineered Heterodimer of Enolase Composed of One Active and One Inactive Subunit

Sims¹,

Menefee²,

Larsen³

et al. 2006

Journal of Molecular Biology

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Enolase is a dimeric enzyme that catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. This reversible dehydration is effected by general acid-base catalysis that involves, principally, Lys345 and Glu211 (numbering system of enolase 1 from yeast). The crystal structure of the inactive E211Q enolase shows that the protein is properly folded. However, K345 variants have, thus far, failed to crystallize. This problem was solved by crystallization of an engineered heterodimer of enolase. The heterodimer was composed of an inactive subunit that has a K345A mutation and an active subunit that has N80D and N126D surface mutations to facilitate ion-exchange chromatographic separation of the three dimeric species. The structure of this heterodimeric variant, in complex with substrate/product, was obtained at 1.85 Å resolution. The structure was compared to a new structure of wild-type enolase obtained from crystals belonging to the same space group. Asymmetric dimers having one subunit exhibiting two of the three active site loops in an open conformation and the other in a conformation having all three loops closed appear in both structures. The K345A subunit of the heterodimer is in the loop-closed conformation; its C α carbon atoms closely match those of the corresponding subunit of wild-type enolase (root-mean-squared deviation of 0.23 Å). The k cat and k cat /K m values of the heterodimer are approximately half those of the N80D/N126D homodimer, which suggests that the subunits in solution are kinetically independent. A comparison of enolase structures obtained from crystals belonging to different space groups suggests that asymmetric dimers can be a consequence of the asymmetric positioning of the subunits within the crystal lattice.

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Structural and mechanistic studies of enolase

Cited by 88 publications

References 26 publications

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Crystal structure of the Escherichia coli RNA degradosome component enolase

Structure and Catalytic Properties of an Engineered Heterodimer of Enolase Composed of One Active and One Inactive Subunit

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