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

Cherfils, Jacqueline; Rosa, R.J. Della; Garel, Marie-Claude; Calvin, Marie-Claude; Rosa, J.; Janin, Joël

doi:10.1016/0022-2836(91)90710-n

Cited by 6 publications

(1 citation statement)

References 10 publications

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“…These activities are catalyzed at the unique active site of BPGM. To date and in spite of recent crystallization of human BPGM (9), no crystallographic data have been obtained for this enzyme. Consequently, the amino acids involved in the active site of human BPGM have been deduced by comparison with the structure of the yeast PGM (8).…”

Section: Introductionmentioning

confidence: 99%

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

Garel¹,

Arous²,

Calvin³

et al. 1994

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

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To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-Dglycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg'-His"'-Gly"1-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly'3 with an Arg or a Lys. In another experiment, we replaced Gly'3 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (n-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly'3 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly'3 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.Some enzymes displaying phosphatase activities such as human prostatic acid phosphatase (1, 2), lysosomal (3) and yeast (4) acid phosphatases, erythrocyte bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4) (5-7), glycolytic phosphoglycerate mutase (PGM; D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) (7,8), and hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (fructose-2,6-bisphosphate 2-phosphatase; D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 3.1.3.46) (1) possess a His residue in their active site, which is transiently phosphorylated during the course of the phosphoryl transfer. It was suggested that most such enzymes involved in the binding of phosphate esters should have at least one cationic group suitably disposed near the active site His in order to facilitate the ori...

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Section: Introductionmentioning

confidence: 99%

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

Garel¹,

Arous²,

Calvin³

et al. 1994

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

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Structural modeling of the human erythrocyte bisphosphoglycerate mutase

et al. 1992

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

Cited by 6 publications

References 10 publications

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

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

Structural modeling of the human erythrocyte bisphosphoglycerate mutase

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

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