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

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-D-glycerate 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 Arg9-His10-Gly11-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 Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 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.

Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89.

Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.

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

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships.

2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.

Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism.

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.

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

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.

The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites.

Enzymes of the glycolytic pathway as well as some ancillary enzymes were studied in normal red cells parasitized with Plasmodium falciparum in culture at varying parasitemias as well as in isolated parasites. The levels of all enzymes except diphosphoglycerate mutase, glucose-6-phosphate dehydrogenase, and adenylate kinase were elevated. Extreme elevations of hexokinase, aldolase, enolase, pyruvate kinase, and adenosine deaminase concentrations were noted. In most cases, electrophoretically distinct bands of enzyme activity were also seen. These findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.

Sequence of the human erythrocyte phosphoglycerate mutase by microsequencer and mass spectrometry.

We have previously reported the isolation in pure form of the human erythrocyte phosphoglycerate mutase isozyme B. We now report the sequence of the whole protein and the identification of its N-terminal blocking group. The protein tryptic peptides of phosphoglycerate mutase isozyme B were isolated by high performance liquid chromatography and their sequence determined by microsequencing. The sequence and the nature of the blocking group of the N-terminal tryptic peptide was shown to be N-acetyl-Ala-Ala-Tyr-Lys by mass spectrometry. Overlaps of the tryptic peptides were obtained by studying the V8 Staphylococcus aureus protease peptides of the aminoethylated phosphoglycerate mutase isozyme B either by microsequencing or by mass spectrometry. The procedure used allowed us to obtain the sequence on a very small amount of material and in a short period of time. Our data agree well with those derived from the cDNA nucleotide sequence described by Sakoda et al. (Sakoda, S., Shanske, S., DiMauro, S., and Schon, E. A. (1988) J. Biol. Chem. 263, 16899-16905). In addition, our data directly indicate that the initiation codon does not introduce a methionine as N-terminal amino acid and allowed the identification of the acetyl N-terminal group.

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.

Rabbit M type phosphoglyceromutase: comparative effects of two thiol reagents antibody reaction and hybridization studies.

1. Treatment of purified rabbit phosphoglyceromutase (M type) with N-ethylmaleimide or with iodoacetamide produces the concurrent loss of phosphoglyceromutase activity with its collateral glycerate-2,3-P2 phosphatase activity. 2. Differences are observed in the protective effect of glycerate-2,3-P2 and of glycolate-2-P against N-ethylmaleimide and iodoacetamide treatments. 3. Specific chicken antibodies obtained by injection of the purified rabbit M type phosphoglyceromutase do not cross-react with the B type but neutralize both rabbit and human M type phosphoglyceromutase. 4. Purified rabbit M type phosphoglyceromutase can hybridize in vitro with the purified human B type or with purified human glycerate-2,3-P2 synthase. 5. Its ability to hybridize with glycerate-2,3-P2 synthase is unchanged after iodoacetamide treatment.

Possibility of prenatal diagnosis of hereditary triose phosphate isomerase deficiency.

Prenatal diagnosis has been performed on umbilical cord blood of an 18 weeks fetus of heterozygous triosephosphate isomerase (TPI) deficient parents. After excluding maternal blood contamination, TPI activity was measured and found to be 60 per cent of the normal mean whereas the value of glucose-6-phosphate dehydrogenase activity was in the normal range of fetal blood. In addition, the analysis of the characteristics of fetal TPI, i.e. Km measurements for glyceraldehyde-3-phosphate, heat stability tests and electrophoretic studies, did not show any evidence of a special form of TPI in fetal blood. These results were consistent with the heterozygous state and were confirmed at birth.

Hereditary triose phosphate isomerase deficiency: seven new homozygous cases.

Seven new homozygous cases of hereditary triosephosphate isomerase (TPI) deficiency have been detected in five unrelated families. Two of the families originate in France, the others from Algeria, Yugoslavia, and Morocco. Only the parents coming from Algeria and Morocco were first cousins. In the other parents no evidence of consanguinity was found. All seven patients exhibited the same symptoms, i.e. hemolytic anemia appearing very early after birth associated with progressive neuromuscular symptoms. Expression of the deficiency is heterogeneous; this had previously been pointed out in the previously reported cases of TPI deficiency. Red cell TPI activity was 3 to 4% of the normal mean in the patients and 50 to 60% in the parents. The latter did not exhibit any clinical symptoms. The levels of red cell glycolytic intermediates and the characteristics of the mutated TPI could be studied in four of the patients only. Substantial increases of red cell dihydroxyacetone phosphate and of fructose 1,6-diphosphate, normal Km of TPI for glyceraldehyde phosphate, and thermoinstability of the enzyme were found. In addition the electrophoretic pattern showed no significant modification of the mobility of the TPI bands, but abnormal decreased staining of the two more anodal bands.

Biochemical and immunological arguments for homology between red cell and liver phosphoglyceromutase isozymes.

Phosphoglyceromutase (2,3-bisphospho-D-glycerate:2- phospho-D-glycerate phosphotransferase, EC 2.7.5.3) from the red cell is compared with the muscle and the liver forms of the enzyme obtained from rabbits. Partially purified samples were used for this study. The electrophoretic mobility of red cell phosphoglyceromutase is completely different from that of muscle and similar to that of liver. The muscle isozyme can hybridize with liver or red blood cell phosphoglyceromutase. N-Ethylmaleimide, an SH group reagent, completely inhibits muscle phosphoglyceromutase activity and slightly inhibits the activity of the enzyme from liver and from red cells. Both liver and red cell isozymes are completely inactivated by heating at 60 degrees C for 15 min, whereas a 25% decrease in inactivity is noted for the muscle enzyme. Finally, chicken antibody directed against human red cell phosphoglyceromutase reacts with the enzyme isolated from rabbit red cells and liver, but not with that obtained from muscle.

Evidence for the presence of a hybrid of phosphoglyceromutase/bisphosphoglyceromutase in the red cells: partial characterization of the hybrid.

Purified phosphoglyceromutase was hybridized in vitro with pure biphosphoglyceromutase . The hybrid showed an electrophoretic mobility identical to that of the intermediate band of red cell phosphoglyceromutase activity in the hemolysate patterns. Electrophoretic tests showed that the partially purified hybrid displayed both bisphosphoglyceromutase and phosphoglyceromutase activities and that the sample contained also a small portion of non-hybrid bisphosphoglyceromutase . By constrast to a non-hybrid mixture of the two purified mutases the hybrid exhibited heat instability of bisphosphoglyceromutase activity and neutralization of phosphoglyceromutase activity by anti- bisphosphoglyceromutase antibody.

[Diphosphoglyceromutase deficiency: new cases associated with erythrocytosis]. / Déficit en diphosphoglycérate mutase: nouveaux cas associés à une polyglobulie.

New cases of diphosphoglyceromutase (DPGM) have been detected, associated with erythrocytosis in two unrelated families. The deficiency appears to be inherited as an autosomal dominant trait. Diphosphoglycerate phosphatase activity paralleled DPGM activity in all the subjects. Three of the latter displayed complete DPGM deficiency with about 0.4% of the normal 2,3-diphosphoglycerate (2,3 DPG) level. The other four showed partial deficiency (about 50% normal mean) with a similar decrease in 2,3-DPG level. The P50 values are in agreement with the red cell 2,3-DPG concentrations. Il all the deficient subjects the ATP level was elevated and the pattern of glycolytic intermediates was disturbed, with an increase in fructose 1,6-diphosphate, triose-phosphates, 3-phosphoglycerate, glucose 1,6-diphosphate, and reduced or normal levels of glucose-6-phosphate and fructose-6-phosphate.

Purification of human erythrocyte phosphoglycerate kinase by dye ligand affinity chromatography.

A new method for the purification of human erythrocyte phosphoglycerate-kinase involving affinity chromatography on dye-ligand media (Red A), in the presence of 3-phosphoglycerate and ATP, is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate, by amino acid analysis and by immunoprecipitation in Ouchterlony plates. Peptide mapping of tryptic digests of the purified enzyme was performed and the immunoneutralization of the enzyme activity evaluated with rabbit antibodies.

Partial characterization of the inactive mutant form of human red cell bisphosphoglyceromutase and comparison with an alkylated form.

The trifunctional enzyme bisphosphoglyceromutase (or diphosphoglycerate mutase) (EC 2.7.5.4) was purified from human red cells and injected into two chickens. Specific anti-bisphosphoglyceromutase antibodies were produced that displayed a single precipitation line on Ouchterlony plates and on immunoelectrophoresis. No cross-reaction of these antibodies was detected with phosphoglyceromutase, the common glycolytic enzyme. Immunoneutralization of bisphosphoglyceromutase and of its two other activities, i.e., bisphosphoglycerate phosphatase and phosphoglyceromutase, was observed for a purified preparation. The anti-bisphosphoglyceromutase antibody reacts with the inactive enzyme present in the hemolysate of a mutant human subject. It also binds bisphosphoglyceromutase inactivated by N-ethylmaleimide, a strong alkylating agent of SH groups. Active bisphosphoglyceromutase is stable at 55 degrees C, whereas the inactive forms of the mutant and of the alkylated hemolysates are thermolabile. These forms can be protected against thermal precipitation by 4 mM 2,3-diphosphoglycerate and 4 mM 3-phosphoglycerate. These findings afford evidence that the binding of the substrates on the bisphosphoglyceromutase molecule is not prevented by alkylation nor by the mutation of the hereditary inactive enzyme.

A new case of phosphoglycerate kinase deficiency: PGK Creteil associated with rhabdomyolysis and lacking hemolytic anemia.

A new case of phosphoglycerate kinase (PGK) deficiency is described. The propositus displayed episodes of rhabdomyolysis crises and acute renal failure but did not exhibit any sign of hemolysis. A severe deficiency in phosphoglycerate kinase was revealed in muscle and was also found in erythrocytes, white cells and platelets. A partial defect in the same enzyme was present in the mother's and the two daughters' erythrocytes, indicating a X-linked recessive genetic transmission of the enzyme defect. In the propositus, erythrocyte ATP concentration was normal, although 2,3-diphosphoglycerate and triose phosphate levels were moderately increased. Lactate production from glucose, in vitro, was close to normal in intact red cells. The partial PGK was characterized by an increased Km for ADP and more especially for ATP, reduced thermostability, and diminished electrophoretic mobility. Lack of this enzyme, which is a key step in the glycolytic process (generation of one molecule of ATP), is thought to be responsible for rhabdomyolysis, a fact that has not been reported previously.