Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies

Calvin, Marie-Claude; Blouquit, Y.; Garel, Marie-Claude; Prehu, Marie-Odette; Cohen-Solal, M.; Rosa, J.; Rosa, R.J. Della

doi:10.1016/0300-9084(90)90029-g

Cited by 10 publications

(15 citation statements)

References 25 publications

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“…Ser 23 Is Involved in the Binding of Both Monophosphoglycerates and 2-Phosphoglycolate-The properties of the S23G variant were very similar to MPGM: the mutase activity was highly effective whereas the synthase reaction was significantly reduced (Tables II-IV). Substitution of Ser by Gly did not affect either of the 2,3-DPG hydrolysis reactions whereas 2-phosphoglycolate stimulated activity was greatly reduced.…”

Section: -Phosphoglycolate-binding Site In Bisphosphoglycerate Mutasementioning

confidence: 95%

“…The native and mutated enzymes were purified by an established procedure (23,33), however, the lysate was not heated prior to chromatography and the first purification step was on an high pressure liquid chromatography column of Fractogel TSK-AF blue at room temperature (24). The purified enzymes were stored in sodium phosphate buffer containing 20% glycerol at Ϫ80°C until used.…”

Section: Methodsmentioning

confidence: 99%

“…The kinetics showed that the replacement of Ser 23 by Ala did not produce any significant change in the enzymatic reactions, however a variant with a Thr residue at position 23 was too unstable to be studied (data not shown).…”

Section: -Phosphoglycolate-binding Site In Bisphosphoglycerate Mutasementioning

confidence: 99%

“…coli-In the human, murine, and rabbit BPGMs, the Cys 22 and Ser 23 residues are strictly conserved (Table I), however, at the position of Ser 23 in BPGMs there is always a Gly in the MPGMs from human muscle and brain and from yeast. In contrast, whereas Cys 22 is conserved in human muscle MPGM, it is substituted by Ser in human brain MPGM and by Thr in …”

Section: Purification Of Wild-type and Mutant Bpgms Expressed In Ementioning

confidence: 99%

“…In addition, the affinity constant for 2-phosphoglycolate was also determined (Tables II-IV). A detailed analysis of all the catalytic activities enabled the functions of Cys 22 , Ser 23 , and Arg 89 in monophosphoglycerates and phosphatase activator binding to be distinguished.…”

Section: -Phosphoglycolate-binding Site In Bisphosphoglycerate Mutasementioning

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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Section: -Phosphoglycolate-binding Site In Bisphosphoglycerate Mutasementioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: -Phosphoglycolate-binding Site In Bisphosphoglycerate Mutasementioning

confidence: 99%

Section: Purification Of Wild-type and Mutant Bpgms Expressed In Ementioning

confidence: 99%

Section: -Phosphoglycolate-binding Site In Bisphosphoglycerate Mutasementioning

confidence: 99%

See 3 more Smart Citations

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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An enzyme-linked immunosorbent assay and reference ranges for bisphosphoglycerate mutase in human erythrocytes

Takubo

Izumi

Tatsumi

et al. 1998

J. Clin. Lab. Anal.

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We established an enzyme-linked immunosorbent assay (ELISA) system for the determination of human bisphosphoglycerate mutase (BPGM) protein content in human erythrocytes using a polyclonal anti-BPGM antibody, we determined reference ranges for BPGM protein content, synthase activity, and specific activity in human erythrocytes. We produced a recombinant human BPGM (rBPGM) by gene manipulation using E. coli and then obtained the polyclonal antibody by immunizing rabbits with purified rBPGM. The reproducibility of the ELISA was in an acceptable range with a coefficient of variation under 1.5%. The ELISA was reliable in the range of 0.1 to 10 ng/mL. The polyclonal anti-rBPGM antibody did not show any cross-reaction with recombinant human B type phosphoglycerate mutase, which is highly homologous to rBPGM. The ELISA was found to be practical for the determination of BPGM protein content in human erythrocytes. The mean BPGM protein content was 56.3 ± 9.7 µg/mL in whole blood (mean ± SD, n = 50). The ELISA can be used to examine various hematologic disorders with abnormal red cell size and cell counts, and to detect BPGM enzymopathy in human erythrocytes.

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Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase

Cherfils¹,

Rosa²,

Garel³

et al. 1991

Journal of Molecular Biology

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Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies

Cited by 10 publications

References 25 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

An enzyme-linked immunosorbent assay and reference ranges for bisphosphoglycerate mutase in human erythrocytes

Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase

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