Bisphosphoglycerate Mutase Research Articles

Isolation, characterization, and structure of a mutant 89 Arg → Cys bisphosphoglycerate mutase: Implication of the active site in the mutation

Abstract Bisphosphoglycerate mutase (EC 5.4.2.4.) is a trifunctional enzyme which displays synthase, mutase, and phosphatase activities. The purification, characterization, and structural study of an abnormal form of the enzyme, isolated from a patient which we reported earlier (Rosa, R., Prehu, M. O., Beuzard, Y., and Rosa, J. (1978) J. Clin. Invest. 62, 907-915), is described. The abnormal enzyme, present at 50% of the level of the normal enzyme as estimated by immunological methods, showed elevated electrophoretic mobility and hybridized with erythrocyte phosphoglycerate mutase (EC 5.4.2.1.) in the same manner as the normal control. The mutant enzyme was unstable at 55 degrees C and could be protected against thermal instability by 0.5 mM glycerate 2,3-bisphoshate but not by either glycerate 3-phosphate or glycolate 2-phosphate. Two of the three functions of the mutant enzyme were distinct from those of the normal protein. The specific activity of the synthase was 0.57% of normal and that of the mutase 4.1%. By contrast, the specific phosphatase activity was not affected by the mutation. However, the phosphatase activity of the mutated protein was markedly less stimulated by glycolate-2-phosphate than that of the control. High performance liquid chromatography analysis of tryptic peptides derived from the mutant enzyme showed an abnormal profile with the absence of two peaks normally containing the T12 and T13 peptides and without the appearance of a supplementary peak. Amino acid sequence and mass spectrometric analysis demonstrated the substitution of Arg----Cys residue in position 89 producing an uncleaved T12-T13 present in the same peak as the T6. Considered together, our data suggest that Arg-89 is located at or near the active site of bisphosphoglycerate mutase and that this residue is probably involved in the binding of monophosphoglycerates.

Comparative effects of two sulfhydryl reagents on the activities of a multifunctional red cell enzyme: Bisphosphoglycerate mutase

The effects of two sulfhydryl reagents on the three activities of bisphosphoglycerate mutase have been compared. Under N-ethylmaleimide treatment all the activities were inhibited except for 60% of the non-stimulated phosphatase. With iodoacetamide the mutase and the stimulated-phosphatase activities were completely inhibited whereas the non-stimulated phosphatase and 60% of the synthase activities were unaffected. 2,3-bisphosphoglycerate protected all the activities of the enzyme against inactivation by the two sulfhydryl reagents whereas 3-phosphoglycerate protected them only against iodoacetamide. 2-phosphoglycolate had an identical effect to that of 3-phosphoglycerate except for its effect on the non-stimulated phosphatase activity, which was slightly enhanced under N-ethylmaleimide treatment.

Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein. A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes. The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each. The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I). Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase. Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M. R., and Pilkis, S. J. (1987) Biochem. Biophys. Res. Commun. 143, 1092-1098). The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods. A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified. However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase.

Bisphosphoglycerate mutase and pyruvate kinase activities during maturation of reticulocytes and ageing of erythrocytes.

An increase in bisphosphoglycerate mutase (BPGM) and a decrease in pyruvate kinase (PK), i.e. a decrease in PK/BPGM ratio, was observed in red cell populations from anemic rats containing 95% down to 3% reticulocytes in blood. Such a ratio has been used to study the fractionation of recticulocytes, according to their degree of maturation, after counter-current distribution of those cell populations in dextranpoly (ethylene glycol) two-phase systems. When applying this procedure to the fractionation according to age of erythrocytes from normal rats, the decrease of PK with cellular age was observed without a significant variation in BPGM activity.

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency.

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.

Changes in enzyme activities involved in the degradation of 1,3-bisphosphoglycerate during erythropoiesis in rat bone marrow.

Rat bone marrow cells have been fractionated by density gradient in Percoll. Differential counting of erythroid cells, haemoglobin concentration and bisphosphoglycerate mutase and phosphoglycerate kinase activities have been determined in cellular fractions. As shown by means of a statistical approach, an increase in bisphosphoglycerate mutase activity and a slight decrease in phosphoglycerate kinase activity is found in erythroid cells as their haemoglobin content increases. Our results suggest that there is a synthesis of 2,3-bisphosphoglycerate during the erythropoietic process which parallels the synthesis of haemoglobin.

A comparison of some of the physical and chemical properties of the phosphocylycerate mutases from human erythrocytes

Summary The molecular weights of both phosphoglycerate mutases from human erythrocytes have been critically examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by exclusion chromatography. The data indicate that native monophospholycerate mutase (mw ≈ 56–57,200) is larger than bisphosphoglycerate mutase (mw ≈ 54–54,400) by approximately 2–3,000 g per mole. Despite differences in mass, amino acid analyses show that each enzyme is strikingly similar in compasition. Thus, the values obtained for two-thirds of the residues in both proteins are either identical or nearly idential. Moderately disparate values, however, are found for lysine, threonine, glutamic acid, proline, glycine, alanine, and leucine. Of the two proteins, the monomutase is the slightly more hydrophobic, while the dimutase is the more acidic. In accord with compositional analyses, density gradient electrofocusing shows that the isoelectric points of bisphosphoglycerate and monophosphoglycerate mutase are 4.9 and 6.2, respectively. The results presented in this paper and elsewhere suggest that an extensive degree of homology exists between both red cell enzymes.

A linear steady-state treatment of enzymatic chains. A mathematical model of glycolysis of human erythrocytes.

A mathematical model of glycolysis in the steady state of human erythrocytes is proposed based on an analytical treatment. Assuming a linear dependence of the enzyme velocities on the substrate concentrations, which is justified for all non-equilibrium enzymes, simple analytical expressions for all metabolites of the glycolysis could be obtained. From a consideration of the NADH/NAD-coupling between the lactate dehydrogenase and the glyceraldehyde-phosphate dehydrogenase reactions a conservation quantity for the concentrations of the oxidized metabolites is derived. The glycolytic flux was shown to be controlled only by the hexokinase and phosphofructokinase. The control strengths for the hexokinase and phosphofructokinase were calculated to be 0.69–0.73 and 0.31–0.27 at pH 7.2 and 0.87–0.9 and 0.13–0.1 at pH 8.2, respectively for various experimental conditions. The metabolites glucose 6-phosphate and fructose 6-phosphate are only influenced by the characteristic times of the hexokinase and phosphofructokinase. All other metabolites and the NADH/NAD ratio depend additionally on the characteristic time the pyruvate kinase. The level of 2,3-bisphosphoglycerate is influenced furthermore by the characteristic time of the bisphosphoglycerate mutase and by the capacity of the 2,3-bisphosphoglycerate phosphatase. The effects of changes in the kinetic properties of enzymes on the concentrations of metabolites may be expressed in form of a control matrix. The enzymes which interact with an outer effector may be identified by means of three guide parameters. The best combination is based on the determination of changes of glucose 6-phosphate, phosphoenol-pyruvate and the total flux. This method is much to be preferred to the usual interpretation of crossover-plots. It is shown that pseudo-crossovers occuring at the glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase and pyruvate kinase are not indicative for an interaction site with an effector. The model was tested in a number of experimental conditions including variation of the pH, Pi concentration, NH4+-ion concentration and temperature for which crossovers have been described. It explains quantitatively the complicated pattern of changes. The conclusions on the properties of the enzymes drawn from the metabolite concentrations are consistent with their behaviour under isolated conditions. It is further shown that under conditions in vitro with lactate accumulation the levels of fructose bisphosphate, triosephosphate and the NADH/NAD ratio are time-dependent.

The phosphorylation of yeast phosphoglycerate mutase

The B-type cofactor-dependent phosphoglycerate mutase (dPGM-B) catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways using 2,3-bisphosphoglycerate as the cofactor. The crystal structures of human dPGM-B bound with citrate were determined in two crystal forms. These structures reveal a dimerization mode conserved in both of dPGM and BPGM (bisphosphoglycerate mutase), based on which a dPGM/BPGM heterodimer structure is proposed. Structural comparison supports that the conformational changes of residues 13–21 and 98–117 determine PGM/BPGM activity differences. The citrate-binding mode suggests a substrate-binding model, consistent with the structure of Escherichia coli dPGM/vanadate complex. A chloride ion was found in the center of the dimer, providing explanation for the contribution of chloride ion to dPGM activities. Based on the structural information, the possible reasons for the deficient human dPGM mutations found in some patients are also discussed.