A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.

Garel, Marie-Claude; Arous, N.; Calvin, Marie-Claude; Craescu, Constantin T.; Rosa, J.; Rosa, R.J. Della

doi:10.1073/pnas.91.9.3593

Cited by 11 publications

(8 citation statements)

References 33 publications

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“…Analysis of the catalytic properties of variants have shown that Arg 89 is specifically involved in the monophosphoglycerate-binding sites (24) as has been reported for a natural human BPGM variant (25)(26)(27). In contrast, we have shown that Gly 13 is specifically involved in the diphosphoglycerate-binding sites and that replacement of Gly 13 by a positively charged amino acid residue greatly activates the phosphatase reaction, whereas the synthase and mutase reactions are greatly reduced (28).…”

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confidence: 79%

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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confidence: 79%

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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“…Increased levels of 2,3‐BPG are present in patients suffering from hypoxemia and anemia, because this facilitates the oxygen release of oxyhemoglobin into tissue 6. In contrast, high 2,3‐BPG levels in individuals with sickle cell anemia are harmful, because 2,3‐BPG favors sickling by facilitating the polymerization of sickle hemoglobin 7–9. Controlling the physiological concentration of 2,3‐BPG could yield a treatment for these diseases.…”

Section: Methodsmentioning

confidence: 99%

Controlling the Oxygenation Level of Hemoglobin by Using a Synthetic Receptor for 2,3‐Bisphosphoglycerate

Zhong

Anslyn

2003

Angewandte Chemie

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Der synthetische Rezeptor 1 mit Guanidinium‐Gruppen und einem Kupfer(II)‐Zentrum bindet 2,3‐Diphosphoglycerat (2,3‐BPG) unter physiologischen Bedingungen effektiv und selektiv (siehe Bild; C=grau, Cu=grün, N=blau, P=gelb, O=rot). Durch seine hohe Affinität verringert der Rezeptor die Konzentration von verfügbarem 2,3‐BPG und nimmt auf diese Weise Einfluss auf die Oxygenierung von Hämoglobin.

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“…A mutation of Gly-14 of hBPGM to Ser did not modify the synthase activity, whereas the mutase and phosphatase activities were 2-fold increased or decreased, respectively. However, replacing Gly-14 with Arg enhanced phosphatase activity by 28.6-fold, whereas synthase and mutase activities were decreased 10-fold (30). In dPGMs, the equivalent residue Ser interacts with the , and marked in green background.…”

Section: Comparison With Dpgmsmentioning

confidence: 99%

Crystal Structure of Human Bisphosphoglycerate Mutase

Wang

Wei

Bian

et al. 2004

Journal of Biological Chemistry

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Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 Å and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and R free of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98 -117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed.Bisphosphoglycerate mutase (BPGM) 1 is an erythrocyte-specific trifunctional enzyme. The main activity is that of synthase (BPGM, EC 5.4.2.4), catalyzing the formation of 2,3-bisphosphoglycerate (2,3-BPG) from 1,3-bisphosphoglycerate (1,3-BPG) (1-3) (see Fig. 1A). The second activity is that of a mutase (phosphoglycerate mutase, EC 5.4.2.1) catalyzing the interconversion between 2-and 3-phosphoglycerate (Fig. 1B) (4). The third activity, as a phosphatase (S-succinylglutathione hydrolase, EC 3.1.3.13), is to catalyze the hydrolysis of 2,3-BPG to 3-or 2-phosphoglycerate and a phosphate (Fig. 1C) (1, 2). The phosphatase reaction can be stimulated by a number of anions including chloride, phosphate, and particularly by 2-phosphoglycolate (5). These three enzymic activities have been found to occur at a unique active site with two different binding sites for the substrates, one for bisphosphoglycerate and another for monophosphoglycerate (6, 7).BPGM regulates the level of 2,3-BPG in human blood cells. In vivo, the concentration of 2,3-BPG is determined by the relative activities of the synthase and phosphatase reactions. In red blood cells, 2,3-BPG is the main allosteric effector of hemoglobin. It shifts the equilibrium between the oxy and deoxy conformations of hemoglobins by preferentially stabilizing the unliganded form. Sickle cell anemia is characterized by polymerization of deoxygenated hemoglobin mutants giving rise to deformed erythrocytes and vaso-occlusive complications. 2,3-BPG has been shown to facilitate this polymerization. The ability to modulate the 2,3-BPG level in vivo wou...

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A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.

Cited by 11 publications

References 33 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

Controlling the Oxygenation Level of Hemoglobin by Using a Synthetic Receptor for 2,3‐Bisphosphoglycerate

Crystal Structure of Human Bisphosphoglycerate Mutase

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