New procedures to measure synthase and phosphatase activities of bisphosphoglycerate mutase. Interest for development of therapeutic drugs

Ravel, Pascale; Garel, Marie-Claude; Toullec, D

doi:10.1016/s0764-4469(99)80083-3

Cited by 2 publications

(4 citation statements)

References 13 publications

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“…Synthase and Mutase Assays-Synthase activity was measured using a simplified method recently published (37). Mutase activity was measured as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

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Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

Ravel

Craescu

Arous

et al. 1997

Journal of Biological Chemistry

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The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates. The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site. BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys 22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the K a value of the activator, whereas it caused no change in other catalytic activities or in the K m values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys 22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding.Kinetic experiments showed that the K a of the cofactor and the K m of 3-PG were affected by the substitution of Ser 23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate. The R89K variant has previously been shown to have a modified K m value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg 89 is specifically involved in monophosphoglycerates binding.CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species.In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM. The specific implication of the Cys 22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.

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“…Synthase and Mutase Assays-Synthase activity was measured using a simplified method recently published (37). Mutase activity was measured as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

“…Phosphatase Assay-Phosphatase activity was measured using a radioactive procedure (37). Following the synthesis of the radioactive substrate [ 33 P]2,3-DPG, the phosphatase reaction was monitored by measuring the radioactivity of monophosphorylated reaction products (P i and 3-PG).…”

Section: Methodsmentioning

confidence: 99%

Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

Ravel

Craescu

Arous

et al. 1997

Journal of Biological Chemistry

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“…Cells (10 9 ) were extracted with 1.5 ml of 2 M PCA and then neutralized with 1.95 ml of 1 M KOH. The 2,3-BPG was then purified by a modification of a procedure published previously (32). Samples were diluted to 50 ml with water and were applied to 4 ϫ 0.8-cm AG 1-X8 anion-exchange columns (Cl Ϫ form; Bio-Rad).…”

Section: 3-bpg Hydrolysis By Dictyostelium Cell Extracts Particulamentioning

confidence: 99%

“…Kinetic parameters were determined by nonlinear regression analysis from substrate-saturation plots by using SigmaPlot. Bisphosphoglycerate synthase was assayed as described previously (32).…”

Section: 3-bpg Hydrolysis By Dictyostelium Cell Extracts Particulamentioning

confidence: 99%

Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport–Luebering glycolytic shunt

Cho

King

Qian

et al. 2008

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

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The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3-bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37°C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4°C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered.2,3-BPG ͉ erythrocyte ͉ glycolysis T he Rapoport-Luebering glycolytic shunt generates and dephosphorylates 2,3-bisphosphoglycerate (2,3-BPG). These reactions have been considered to be catalyzed by a single 2,3-BPG synthase/2-phosphatase (BPGM) (1, 2). This enzyme has mutase activity that converts the glycolytic intermediate, 1,3-BPG, to 2,3-BPG. BPGM also acts as a phosphatase, converting 2,3-BPG to 3-phosphoglycerate (3-PG), which then reenters the main glycolytic pathway. This has been the textbook perception of this metabolic pathway for Ͼ25 years (3).Metabolic flux through the Rapoport-Luebering shunt carries an energetic cost for the cell because it bypasses the ATPgenerating phosphoglycerate kinase. Nevertheless, in vertebrates and lower eukaryotes, 2,3-BPG fulfills an essential role in glycolysis by priming the phosphoglycerate mutase reaction that converts 3-PG to 2-PG (4). More recently, experiments in Dictyostelium have suggested that 2,3-BPG, through an unknown mechanism, provides a molecular link between the turnover of phosphorylated inositol derivatives and glycolytic flux (5). Nonetheless, the most well studied function for 2,3-BPG is its regulation of whole-body oxygen homeostasis; 2,3-BPG executes this role because it is the main allosteri...

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New procedures to measure synthase and phosphatase activities of bisphosphoglycerate mutase. Interest for development of therapeutic drugs

Cited by 2 publications

References 13 publications

Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport–Luebering glycolytic shunt

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