UNIPROT:P00790 - FACTA Search

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Query: UNIPROT:P00790 (PGA)
2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The high oxygen affinity of fetal blood in rabbits is due to a very low concentration of 2,3-diphosphoglycerate (2,3-DPG) in the red cells. In order to gather informations on the factors responsible for this characteristic we have studied synthesis and break-down of 2,3-DPG in fetal and adult rabbit red cells in vitro and examined possible regulative pathways which may lead to the low 2,3-DPG concentration in vivo. 2. Under conditions where 2,3-DPG and 3-phosphoglycerate (3-PGA) accumulate in adult erythrocytes, i.e. in a solution containing inosine, pyruvate and inorganic phosphate, the amount of 2,3-DPG synthetized in fetal red cells was only 40% of the adult value and 3-PGA was not measurable. Upon inhibition of enolase by NaF, however, both 2,3-DPG and 3-PGA increased to a similar extent in fetal and adult red cells. These findings point towards differences in the pyruvate kinase (PK) reaction which is one of the rate limiting steps of glycolysis. Direct measurements revealed an over tenfold higher PK activity in fetal compared to adult red cells. This higher activity of PK will lead to a decreased concentration of 3-PGA with a consecutive fall in 2,3-DPG concentration. 3. Other factors, like a decreased glucose utilization, a decreased activity of 2,3-DPG mutase or an increased 2,3-DPG phosphatase activity could be excluded as a cause for the low 2,3-DPG concentration in fetal red blood cells. The same holds for extraerythrocytic factors like glucose concentration or pH value in fetal blood. 4. During the postnatal development of rabbits the PK activity decreased. 50 days after birth, PK activity was 20% of the fetal value but still somewhat higher than in adult erythrocytes. This change is paralleled by an increase in 2,3-DPG concentration and half saturation oxygen pressure. With respect to the synthesis of 2,3-DPG and ATP, the fetal rabbit red cell is comparable to hereditary high PK activity in human erythrocytes.
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PMID:High pyruvate kinase activity causes low concentration of 2,3-diphosphoglycerate in fetal rabbit red cells. 2 78

Ten tryptophan residues per one protein molecule were found to be present in the enolase from human and swine muscle. In Tris buffer, N-bromosuccinimide (NBS) inactivated the enolases after oxidation of all 10 tryptophan residues. The presence of 2-phosphoglycerate (2-PGA) partially protected the activity, and in the presence of 2-PGA together with Mg2+ full protection was observed. In phosphate buffer, only 6 tryptophan residues could be oxidized, but the enzyme was fully inactivated. 2-PGA made possible the oxidation of all 10 tryptophan residues, concomitant with full inactivation. In either case, Mg2+ had no effect. The Km values and pH optima were the same for the native and partially NBS-modified enolases.
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PMID:The reaction of N-bromosuccinimide with enolase. 61 Feb 83

The equilibrium mixture of yeast enolase with substrate, 2-phospho-D-glycerate (2-PGA), and product, phosphoenolpyruvate (P-enolpyruvate), has been crystallized from solutions of poly(ethylene glycol) (PEG) at pH 8.0. Crystals belong to the space group C2 and have unit cell dimensions a = 121.9 A, b = 73.2 A, c = 93.9 A, and beta = 93.3 degrees. The crystals have one dimer per asymmetric unit. Crystals of the equilibrium mixture and of the enolase complex of phosphonoacetohydroxamate (PhAH) are isomorphous, and the structure of the former complex was solved from the coordinates of enolase-(Mg2+)2-PhAH [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. The current crystallographic R-factor is 17.7% for all recorded data (92% complete) to 1.8 A resolution. The electron density map is unambiguous with respect to the positions and liganding of both magnesium ions and with respect to the stereochemistry of substrate/product binding. Both magnesium ions are complexed to functional groups of the substrate/product. The higher affinity Mg2+ coordinates to the carboxylate side chains of Asp 246, Glu 295, and Asp 320, both carboxylate oxygens of the substrate/product, and a water molecule. One of the carboxylate oxygens of the substrate/product also coordinates to the lower affinity Mg2+-thus forming a mu-carboxylato bridge. The other ligands of the second Mg2+ are a phosphoryl oxygen of the substrate/product, two water molecules, and the carbonyl and gamma-oxygens of Ser 39 from the active site loop. The intricate coordination of both magnesium ions to the carboxylate group suggests that both metal ions participate in stabilizing negative charge in the carbanion (aci-carboxylate) intermediate. The epsilon-amino group of Lys 345 is positioned to serve as the base in the forward reaction whereas the carboxylate side chain of Glu 211 is positioned to interact with the 3-OH of 2-PGA. The structure provides a candid view of the catalytic machinery of enolase.
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PMID:A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 A resolution. 860 83

High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.
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PMID:Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants. 863 1

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pHo) for activity is 8.1-8.2, and the Km for 2-PGA is approximately 0.67 mM. Using the dimeric Calpha structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86-96) and the loop between helix L and strand 1 (HL-S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL-S1 loop is truncated by 4-6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL-S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.
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PMID:A model of the quaternary structure of enolases, based on structural and evolutionary analysis of the octameric enolase from Bacillus subtilis. 998 32

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as alphaalpha-(pI = 6.5), alphagamma- (pI = 5.6), and gammagamma-enolase (pI = 5.2). The pI of purified gammagamma-enolase was also 5.2. The gammagamma-enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55 degrees C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%-80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, gammagamma-enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The Km values for 2-PGA, PEP, and Mg2+ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to Km values of other vertebrate enolases. The amino acid composition of pig gammagamma-enolase, as determined by amino acid analysis, shows strong similarity to the compositions of gammagamma-enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig gammagamma-enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig gammagamma-enolase and the other gammagamma-enolases are almost identical. Li+ proved to be a noncompetitive inhibitor with either 2-PGA or Mg2+ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2(1). An Rsymm <5% was obtained for data between 50 and 3.65 A, but was a disappointing 30% for data between 3.65 and 3.10 A, indicating crystal disorder.
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PMID:Purification and properties of gammagamma-enolase from pig brain. 1007 35

There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle. However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously. In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor [Duquerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase this residue has been mutated to Ala. The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli. The purified mutant protein is folded properly as indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction. We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site. This proposal is consistent with the catalytic mechanism of yeast enolase. Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C(2) proton from 2-PGA. Metal binding studies support the above proposal. Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase. The factor of 3 in binding is reasonable for the contribution to binding strength of a single nondominant ligand in a chelate [Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094-10100. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281. Cha et al. (1994) J. Biol. Chem. 269, 2687-2694].
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PMID:Role of His159 in yeast enolase catalysis. 1050 18

Enolase from rabbit muscle (betabeta-enolase) is inactivated by NaClO(4). Enolase free of divalent cations is more susceptible to inactivation by NaClO(4) than is enolase in the presence of Mg(2+). We find that substrate protects apo-enolase against inactivation, indicating that substrate can bind to enolase in the absence of a divalent cation. This binding is not due to contamination by trace levels of divalent cations since (1) it occurs even in the presence of EDTA or EGTA and (2) metal analysis by ICP (inductively coupled plasma) mass spectrometry did not reveal sufficient contamination to account for the protection. The binding of PGA to apo-enolase did require Na(+). When TMAClO(4) was used instead of NaClO(4), there was no protection by PGA. Protection was restored when TMAClO(4) plus NaCl were used. The inactivation of apo-enolase by NaClO(4) is due to dissociation into inactive monomers. We conclude that Na(+) binds to apo-enolase, permitting substrate to then bind. Of the three known Me(2+) binding sites on enolase, we believe the most likely binding site for Na(+) is the carboxylate cluster of site 1, the highest affinity site of enolase.
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PMID:The binding of Na(+) to apo-enolase permits the binding of substrate. 1066 92

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) Biochemistry 35, 4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 17O-labeled forms of phosphoenolpyruvate show that Mn(2+), bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of 2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of 2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn(2+) gives lower maximal activity than Mg(2+), apparently due to slow release of Zn(2+) from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn(2+) from the enzyme.
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PMID:Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme. 1143 70

Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala. In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site. With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously [J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351-355]. Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining. Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate. Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities. The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA. The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH). The structure was solved and refined at a resolution of 2.1 A. The structure confirms the conjecture that the active-site flap is opened in the mutant protein. PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex. Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein. His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center. A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site.
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PMID:Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase. 1205 65

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