Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: in vivo kinetic characterization of 2,3-bisphosphoglycerate synthase/phosphatase using 13C and 31P NMR. (1/55)

This is the first in a series of three papers [see also Mulquiney and Kuchel (1999) Biochem. J. 342, 579-594; Mulquiney and Kuchel (1999) Biochem. J. 342, 595-602] that present a detailed mathematical model of erythrocyte metabolism which explains the regulation and control of 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is a modulator of haemoglobin oxygen affinity and hence plays an important role in blood oxygen transport and delivery. This paper presents an in vivo kinetic characterization of 2,3-BPG synthase/phosphatase (BPGS/P), the enzyme that catalyses both the synthesis and degradation of 2,3-BPG. Much previous work had indicated that the behaviour of this enzyme in vitro is markedly different from that in vivo. (13)C and (31)P NMR were used to monitor the time courses of selected metabolites when erythrocytes were incubated with or without [U-(13)C]glucose. Simulations of the experimental time courses were then made. By iteratively changing the parameters of the BPGS/P part of the model until a good match between the NMR-derived data and simulations were achieved, it was possible to characterize BPGS/P kinetically in vivo. This work revealed that: (1) the pH-dependence of the synthase activity results largely from a strong co-operative inhibition of the synthase activity by protons; (2) 3-phosphoglycerate and 2-phosphoglycerate are much weaker inhibitors of 2,3-BPG phosphatase in vivo than in vitro; (3) the K(m) of BPGS/P for 2,3-BPG is significantly higher than that measured in vitro; (4) the maximal activity of the phosphatase in vivo is approximately twice that in vitro, when P(i) is the sole activator (second substrate); and (5) 2-phosphoglycollate appears to play no role in the activation of the phosphatase in vivo. Using the newly determined kinetic parameters, the percentage of glycolytic carbon flux that passes through the 2, 3-BPG shunt in the normal in vivo steady state was estimated to be 19%.  (+info)

Phosphocreatine-dependent protein phosphorylation in rat skeletal muscle. (2/55)

Phosphocreatine (PCr) was found to alter the phosphorylation state of two proteins of apparent molecular masses 18 and 29 kDa in dialysed cell-free extracts of rat skeletal muscle in the presence of [gamma-32P]ATP. The 29 kDa protein was identified as phosphoglycerate mutase (PGM), phosphorylated at the active-site histidine residue by 2,3-bisphosphoglycerate (2,3-biPG). 2,3-biPG labelling from [gamma-32P]ATP occurred through the concerted action of phosphoglycerate kinase and 2,3-bisphosphoglycerate mutase. PCr-dependent labelling, which required creatine kinase, resulted from a shift in the phosphoglycerate kinase equilibrium towards 1,3-bisphosphoglycerate (1,3-biPG) synthesis, ultimately resulting in an increase in available [2-32P]2,3-biPG. The maximal catalytic activity of PGM was unaffected by PCr. The 18 kDa protein was transiently phosphorylated at a histidine residue, probably by 1,3-biPG. No proteins of this monomeric molecular mass are known to bind 1,3-biPG, suggesting that the 18 kDa protein is an undescribed phosphoenzyme intermediate. Previous observations of 2- and 3-phosphoglycerate-dependent protein phosphorylation in cytosolic extracts [Ueda & Plagens (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1229-1233; Pek, Usami, Bilir, Fischer-Bovenkerk & Ueda (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4294-4298], attributed to the action of novel kinases, are likely to represent phosphoenzyme intermediates labelled by bisphosphorylated metabolites in a similar manner.  (+info)

Immunocytochemical localization of glycolytic and fermentative enzymes in Zymomonas mobilis. (3/55)

Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.  (+info)

A single MEF-2 site is a major positive regulatory element required for transcription of the muscle-specific subunit of the human phosphoglycerate mutase gene in skeletal and cardiac muscle cells. (4/55)

In order to analyze the transcriptional regulation of the muscle-specific subunit of the human phosphoglycerate mutase (PGAM-M) gene, chimeric genes composed of the upstream region of the PGAM-M gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into C2C12 skeletal myocytes, primary cultured cardiac muscle cells, and C3H10T1/2 fibroblasts. The expression of chimeric reporter genes was restricted in skeletal and cardiac muscle cells. In C2C12 myotubes and primary cultured cardiac muscle cells, the segment between nucleotides -165 and +41 relative to the transcription initiation site was sufficient to confer maximal CAT activity. This region contains two E boxes and one MEF-2 motif. Deletion and substitution mutation analysis showed that a single MEF-2 motif but not the E boxes had a substantial effect on skeletal and cardiac muscle-specific enhancer activity and that the cardiac muscle-specific negative regulatory region was located between nucleotides -505 and -165. When the PGAM-M gene constructs were cotransfected with MyoD into C3H10T1/2, the profile of CAT activity was similar to that observed in C2C12 myotubes. Gel mobility shift analysis revealed that when the nuclear extracts from skeletal and cardiac muscle cells were used, the PGAM-M MEF-2 site generated the specific band that was inhibited by unlabeled PGAM-M MEF-2 and muscle creatine kinase MEF-2 oligomers but not by a mutant PGAM-M MEF-2 oligomer. These observations define the PGAM-M enhancer as the only cardiac- and skeletal-muscle-specific enhancer characterized thus far that is mainly activated through MEF-2.  (+info)

Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181. (5/55)

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.  (+info)

Compound heterozygosity in a complete erythrocyte bisphosphoglycerate mutase deficiency. (6/55)

Erythrocyte bisphosphoglycerate mutase (BPGM) deficiency is a rare disease associated with a decrease in 2,3-diphosphoglycerate concentration. A complete BPGM deficiency was described in 1978 by Rosa et al (J Clin Invest 62:907, 1978) and was shown to be associated with 30% to 50% of an inactive enzyme detectable by specific antibodies and resulting from an 89 Arg-->Cys substitution. The propositus' three sisters exhibited the same phenotype, while his two children had an intermediate phenotype. Samples from the family were examined using polymerase chain reaction and allele-specific oligonucleotide hybridization and sequencing techniques. Amplification of erythrocyte total RNA from the propositus' sister around the 89 mutation indicated the presence of two forms of messenger RNAs, a major form with the 89 Arg-->Cys mutation and a minor form with a normal sequence. Sequence studies of the propositus' DNA samples indicated heterozygosity at locus 89 and another heterozygosity with the deletion of nucleotide C 205 or C 206. Therefore, the total BPGM deficiency results from a genetic compound with one allele coding for an inactive enzyme (mutation BPGM Creteil I) and the other bearing a frameshift mutation (mutation BPGM Creteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Creteil I mutation.  (+info)

Study of a kindred with partial deficiency of red cell 2,3-diphosphoglycerate mutase (2,3-DPGM) and compensated hemolysis. (7/55)

A kindred with partial deficiency of red cell 2,3-diphosphoglycerate mutase (2,3-DPGM) was studied. The propositus presented with indirect hyperbilirubinemia, normal hemoglobin (15.8 g/dl), and elevated reticulocyte count (4.6%). The red cell 51Cr survival was decreased (tau1/2 16 days). Incubated osmotic fragility was normal; autohemolysis was increased and corrected with glucose and ATP. The P50 was 18.5 mm Hg (normal 25.5 +/- 3), but the stability, electrophoresis, and fingerprinting of hemoglobin were normal. The concentration of 2,3-diphosphoglycerate (2,3-DPG) was reduced to 43% of normal. Red cell 2,3-DPGM was decreased to 59% of normal; 2,3-DPG phosphatase was similarly decreased. All red cell glycolytic and hexose monophosphate shunt enzymes, glycolytic intermediates other than 2,3-DPG, and glucose consumption and lactate production were normal. Five family members showed similar hematologic findings. The deficiency appears to be secondary to decreased enzyme synthesis and to be inherited as an autosomal dominant trait in this family. Partial deficiency of 2,3-DPGM should now be considered in the differential diagnosis of compensated hemolysis associated with increased oxygen affinity.  (+info)

Red cell diphosphoglycerate mutase. Immunochemical studies in vertebrate red cells, including a human variant lacking 2,3-DPG. (8/55)

Diphosphoglycerate mutase (DPGM) was purified to homogeneity from human erythrocytes. The enzyme and Freund adjuvant were injected into chickens and yielded a monospecific precipitating antibody. Radial immunodiffusion with this antibody was used to measure the amount of DPGM in hemolysates from human adult and cord red cells. Dog, rabbit, rat, chicken, and goat red cells all had DPGM during the neonatal period, but goat adult red cells had no detectable enzyme. Single bands with no spurs were present on Ouchterlony plates in which human hemolysate was placed adjacent to hemolysates from the other species tested. The amount of human red cell DPGM did not differ between young and old cells separated by centrifugation. Red cells from a patient with a DPGM genetic variant who had erythrocytosis and no detectable enzyme activity contained a reduced amount of DPGM as determined by radial immunodiffusion. The abnormal DPGM differed from normal by immunoelectrophoresis and in stability as measured by the amount of crossreacting material in young versus old erythrocytes.  (+info)

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