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Background: Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown., Methods: Pregnant mice were subjected to acute or chronic hypoxia (12.5% O 2 ) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry., Results: We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation., Conclusions: This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR., Funding: This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19., Competing Interests: SS, TH, LF, LB, RP, OG, DH, SB, AB, TM, ND No competing interests declared, MK, TB is the Head of the Israel Society for Maternal Fetal Medicine (ISMFM). The author has no other competing interests to declare, MN Michal Neeman has received the Killcorona grant from the Israel Science Foundation and the Collaborative Grant in Biomedical Research from Weizmann - Ichilov. The author is also the elected President of European Society of Molecular Imaging. The author has no other competing interests to declare, (© 2024, Stroganov et al.)

Erythrocytosis is associated with increased red blood cell mass and can be either congenital or acquired. Congenital secondary causes are rare and include germline variants increasing haemoglobin (Hb)-oxygen affinity (e.g., Hb or bisphosphoglycerate mutase (BPGM) variants) or affecting oxygen-sensing pathway proteins. Here, we describe five adults from three kindreds with erythrocytosis associated with heterozygosity for BPGM variants, including one novel. Functional analyses showed partial BPGM deficiency, reduced 2,3-bisphosphoglycerate levels and/or increased Hb-oxygen affinity. We also review currently known BPGM variants. This study contributes to raising awareness of BPGM variants, and in particular that heterozygosity for BPGM deficiency may already manifest clinically., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Erythrocytosis, or increased red cell mass, may be labeled as primary or secondary, depending on whether the molecular defect is intrinsic to the red blood cells/their precursors or extrinsic to them, the latter being typically associated with elevated erythropoietin (EPO) levels. Inherited/congenital erythrocytosis (CE) of both primary and secondary types is increasingly recognized as the cause in many patients in whom acquired, especially neoplastic causes have been excluded. During the past two decades, the underlying molecular mechanisms of CE are increasingly getting unraveled. Gain-in-function mutations in the erythropoietin receptor gene were among the first to be characterized in a disorder termed primary familial and congenital polycythemia. Another set of mutations affect the components of the oxygen-sensing pathway. Under normoxic conditions, the hypoxia-inducible factor (HIF), upon hydroxylation by the prolyl-4-hydroxylase domain protein 2 (PHD2) enzyme, is degraded by the von Hippel-Lindau protein. In hypoxic conditions, failure of prolyl hydroxylation leads to stabilization of HIF and activation of the EPO gene. CE has been found to be caused by loss-of-function mutations in VHL and PHD2/EGLN1 as well as gain-of-function mutations in HIF-2α (EPAS1), all resulting in constitutive activation of EPO signaling. Apart from these, globin gene mutations leading to formation of high oxygen affinity hemoglobins also cause CE. Rarely, bisphosphoglycerate mutate mutations, affecting the 2,3-bisphosphoglycerate levels, can increase the oxygen affinity of hemoglobin and cause CE. This narrative review examines the current mutational spectrum of CE and the distinctive pathogenetic mechanisms that give rise to this increasingly recognized condition in various parts of the world., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Oxygen is vital for oocyte maturation; however, oxygen regulation within ovarian follicles is not fully understood. Hemoglobin is abundant within the in vivo matured oocyte, indicating potential function as an oxygen regulator. However, hemoglobin is significantly reduced following in vitro maturation (IVM). The molecule 2,3-bisphosphoglycerate (2,3-BPG) is essential in red blood cells, facilitating release of oxygen from hemoglobin. Towards understanding the role of 2,3-BPG in the oocyte, we characterized gene expression and protein abundance of bisphosphoglycerate mutase (Bpgm), which synthesizes 2,3-BPG, and whether this is altered under low oxygen or hemoglobin addition during IVM., Methods: Hemoglobin and Bpgm expression within in vivo matured human cumulus cells and mouse cumulus-oocyte complexes (COCs) were evaluated to determine physiological levels of Bpgm. During IVM, Bpgm gene expression and protein abundance were analyzed in the presence or absence of low oxygen (2% and 5% oxygen) or exogenous hemoglobin., Results: The expression of Bpgm was significantly lower than hemoglobin when mouse COCs were matured in vivo. Following IVM at 20% oxygen, Bpgm gene expression and protein abundance were significantly higher compared to in vivo. At 2% oxygen, Bpgm was significantly higher compared to 20% oxygen, while exogenous hemoglobin resulted in significantly lower Bpgm in the COC., Conclusion: Hemoglobin and 2,3-BPG may play a role within the maturing COC. This study shows that IVM increases Bpgm within COCs compared to in vivo. Decreasing oxygen concentration and the addition of hemoglobin altered Bpgm, albeit not to levels observed in vivo.

The replication cycle and pathogenesis of the Plasmodium malarial parasite involves rapid expansion in red blood cells (RBCs), and variants of certain RBC-specific proteins protect against malaria in humans. In RBCs, bisphosphoglycerate mutase (BPGM) acts as a key allosteric regulator of hemoglobin/oxyhemoglobin. We demonstrate here that a loss-of-function mutation in the murine Bpgm (Bpgm L166P ) gene confers protection against both Plasmodium-induced cerebral malaria and blood-stage malaria. The malaria protection seen in Bpgm L166P mutant mice is associated with reduced blood parasitemia levels, milder clinical symptoms, and increased survival. The protective effect of Bpgm L166P involves a dual mechanism that enhances the host's stress erythroid response to Plasmodium-driven RBC loss and simultaneously alters the intracellular milieu of the RBCs, including increased oxyhemoglobin and reduced energy metabolism, reducing Plasmodium maturation, and replication. Overall, our study highlights the importance of BPGM as a regulator of hemoglobin/oxyhemoglobin in malaria pathogenesis and suggests a new potential malaria therapeutic target., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Clinical use of doxorubicin (DOX) in cancer therapy is limited by its dose-dependent cardiotoxicity. But molecular mechanisms underlying this phenomenon have not been well defined. This study was to investigate the effect of DOX on the changes of global genomics in hearts. Acute cardiotoxicity was induced by giving C57BL/6J mice a single intraperitoneal injection of DOX (15 mg/kg). Cardiac function and apoptosis were monitored using echocardiography and TUNEL assay at days 1, 3 and 5. Myocardial glucose and ATP levels were measured. Microarray assays were used to screen gene expression profiles in the hearts at day 5, and the results were confirmed with qPCR analysis. DOX administration caused decreased cardiac function, increased cardiomyocyte apoptosis and decreased glucose and ATP levels. Microarrays showed 747 up-regulated genes and 438 down-regulated genes involved in seven main functional categories. Among them, metabolic pathway was the most affected by DOX. Several key genes, including 2,3-bisphosphoglycerate mutase (Bpgm), hexokinase 2, pyruvate dehydrogenase kinase, isoenzyme 4 and fructose-2,6-bisphosphate 2-phosphatase, are closely related to glucose metabolism. Gene co-expression networks suggested the core role of Bpgm in DOX cardiomyopathy. These results obtained in mice were further confirmed in cultured cardiomyocytes. In conclusion, genes involved in glucose metabolism, especially Bpgm, may play a central role in the pathogenesis of DOX-induced cardiotoxicity.

Hereditary erythrocytosis, thrombocytosis, and neutrophilia are rare inherited syndromes which exhibit Mendelian inheritance. Some patients with primary hereditary erythrocytosis exhibit a mutation in the erythropoietin receptor (EPOR) which is associated with low serum erythropoietin (EPO) levels. Secondary congenital erythrocytosis may be characterized by normal or high serum EPO levels, and is related to high oxygen affinity haemoglobin variants, mutation of the enzyme biphosphoglycerate mutase (BPGM), or defects in components of the oxygen-sensing pathway. Hereditary thrombocytosis was first linked to mutations in genes encoding thrombopoietin (THPO) or the thrombopoietin receptor, MPL. More recently, germline mutations in JAK2, distinct from JAK2 V617F, and mutation of the gelsolin gene, were uncovered in several pedigrees of hereditary thrombocytosis. Hereditary neutrophilia has been described in one family with an activating germline mutation in CSF3R. The mutational basis for most hereditary myeloproliferative disorders has yet to be identified., (Copyright © 2014. Published by Elsevier Ltd.)

Bisphosphoglycerate mutase (BPGM) catalyses the formation of 2,3 bisphosphoglycerate (BPG) a ligand of haemoglobin. BPG facilitates liberation of oxygen from haemoglobin at low oxygen tension enabling efficient delivery of oxygen to tissues. We describe expression of BPGM in mouse labyrinthine trophoblasts, located at the maternal-placental interface. Expression is lower in placentae of igf2(+/-) knockout mice, a widely used model of growth restriction, compared to wild type placentae. Circulating maternal BPG increased throughout gestation but this increase was less in wt mothers carrying igf2(+/-) pups than in those carrying exclusively wt pups. This reduction was observed well before term and may contribute to the low birth weight of igf2(+/-) pups. Strikingly, we also measured reductions of fetal and placental weight in wt littermates of igf2(+/-) pups compared to pups developing in an exclusively wt environment. These data suggest that placental expression of BPGM can influence maternal BPG concentrations and supports a hypothesis under which BPG synthesized in the placenta may act on maternal haemoglobin to enhance delivery of oxygen to the developing fetus.

Glycolysis is one of the principle pathways of ATP generation in cells and is present in all cell tissues; in erythrocytes, glycolysis is the only pathway for ATP synthesis since mature red cells lack the internal structures necessary to produce the energy vital for life. Red cell deficiencies have been detected in all erythrocyte glycolytic pathways, although their frequencies differ owing to diverse causes, such as the affected enzyme and severity of clinical manifestations. The number of enzyme deficiencies known is endless. The most frequent glycolysis abnormality is pyruvate kinase deficiency, since around 500 cases are known, the first of which was reported in 1961. However, only approximately 200 cases were due to mutations. In contrast, only one case of phosphoglycerate mutase BB type mutation, described in 2003, has been detected. Most mutations are located in the coding sequences of genes, while others, missense, deletions, insertions, splice defects, premature stop codons and promoter mutations, are also frequent. Understanding of the crystal structure of enzymes permits molecular modelling studies which, in turn, reveal how mutations can affect enzyme structure and function.

2,3-Bisphosphoglycerate mutase (2,3-BPGM), an erythroid-expressed enzyme, synthesises 2,3-bisphosphoglycerate (2,3-BPG), the allosteric modulator of haemoglobin. This ligand has a higher affinity for adult haemoglobin than for fetal haemoglobin and differential binding of it facilitates transfer of oxygen between adult and fetal blood by lowering the affinity of adult haemoglobin for oxygen. This paper reports the discovery that 2,3-BPGM is synthesised in non-erythroid cells of the human placenta. Western blot analysis of placental extracts revealed high levels of 2,3-BPGM in the human placenta. Immunohistochemical staining and in situ hybridisation experiments indicated that abundant 2,3-BPGM is present in the syncytiotrophoblast layer of the placental villi at the feto-maternal interface. A cytochemical staining technique showed that the placental 2,3-BPGM is active, indicating that 2,3-BPG is synthesised in the outermost cells of the placenta. These observations demonstrate an unexpected and abundant presence of an enzyme key to oxygen release from adult haemoglobin, at the interface between maternal and fetal circulations.

A 28-year-old asymptomatic male of Iranian Jewish (Meshadi) heritage was found on routine exam to have an erythrocytosis (RBC = 6.22 x 10(12)/l, Hgb = 19.2 g/dl, Hct = 58.9%). Splenomegaly was absent on physical exam. There was no family history of erythrocytosis. His oxygen dissociation curve was left-shifted with a p50 of 19 mmHg (normal = 25-32 mmHg). Hemoglobin electrophoresis showed no abnormalities. DNA sequencing of the hemoglobin beta globin gene and both alpha globin genes did not reveal a mutation. A 2,3-bisphosphoglycerate (BPG) level was markedly decreased at 0.3 micromol/g Hb (normal = 11.4-19.4 micromol/g Hb). The patient's bisphosphoglycerate mutase (BPGM) enzyme activity was also markedly decreased at 0.16 IU/g Hb (normal = 4.13-5.43 IU/g Hb). A red cell enzyme panel revealed a markedly decreased G-6-PD level (0.3 U/g Hb, normal = 8.6-18.6 U/g Hb). His parents and a brother were also available for evaluation. Both parents showed normal 2,3-BPG levels but BPGM activity approximately 50% of normal. Paradoxically, the brother showed normal BPGM activity but a slightly decreased 2,3-BPG level. All family members had markedly decreased G-6-PD activity. DNA sequencing of the BPGM gene showed the propositus to be homozygous for 185 G-->A, Arg 62 Gln in exon 2. Thus, the erythrocytosis in this patient is secondary to low 2,3-BPG levels, due to a deficiency in BPG mutase. This appears due to consanguinity within this family., (Copyright 2004 Wiley-Liss, Inc.)

Objectives: The effects of triiodothyronine (T(3)) and hypoxia on 2,3-bisphosphoglycerate (2,3-BPG) studied in vitro are unclear. To clarify these effects we selected a more physiologic approach: the in vivo study in rabbits. We also present the changes produced by T(3) and hypoxia on phosphoglycerate mutase (PGAM), which requires 2,3-BPG as a cofactor, and 2,3-BPG synthase (BPGS), the enzyme responsible for 2,3-BPG synthesis in erythroblasts and reticulocytes., Methods: Hyperthyroidism was induced by daily T(3) injection (250 microg/kg), hypoxia by a mixture of 90% nitrogen and 10% oxygen and hypothyroidism by propylthiouracil (PTU) added to drinking water., Results: Both T(3) administration and hypoxic conditions increased 2,3-BPG levels and BPGS mRNA levels and activity in erythroblasts but not in reticulocytes. Unlike BPGS, both PGAM mRNA levels and activity were increased in erythroblasts and reticulocytes under hyperthyrodism and hypoxia. The antihormone PTU produced opposite effects to T(3)., Conclusion: The results presented here suggest that both hyperthyroidism and hypoxia modulate in vivo red cell 2,3-BPG content by changes in the expression of BPGS. Similarly, the changes in PGAM activity are also explained by changes in its expression., (Copyright (c) 2004 S. Karger AG, Basel.)

Red blood cells can only fulfil their functions over the normal period of approximately 120 days with 1.7 x 10(5) circulatory cycles efficiently if they withstand external and internal loads. This requires ATP and redox equivalents, which have to be permanently regenerated by the energy and redox metabolism. These pathways are necessary to maintain the biconcave shape of the cells, their specific intracellular cation concentrations, the reduced state of hemoglobin with a divalent iron and the sulfhydryl groups of enzymes, glutathione and membrane components. If an enzyme deficiency of one of these metabolic pathways limits the ATP and/or NADPH production, distinct membrane alterations result causing a removal of the damaged cells by the monocyte-macrophage system. Most metabolic needs of erythrocytes are covered by glycolysis, the oxidative pentose phosphate pathway (OPPP), the glutathione cycle, nucleotide metabolism and MetHb reductase. Hereditary enzyme deficiencies of all these pathways have been identified; those that cause non-spherocytic hemolytic anemia are listed in Table 4. Their frequencies differ markedly both with respect to the affected enzyme and geographic distribution. Glucose-6-phosphate dehydrogenase enzymopathies (G6PD) are with more than 400 million cases by far the most common deficiency. The highest gene frequency has been found with 0.7 among Kurdish Jews. G6PD deficiencies are furthermore prevalent with frequencies of about 0.1 among Africans, Black Americans, and populations of Mediterranean countries and South East Asia. In Middle and Northern Europe the frequency of G6PD is much lower, and with approximately 0.0005, comparable with the frequency of pyruvate kinase (PK) enzymopathies, the most frequent enzyme deficiency in glycolysis in this area (Luzzatto, 1987; Beutler and Kuhl, 1990). The relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction in red blood cells and other tissues depend on several factors: on the importance of the affected enzyme; its expression rate; the stability of the mutant enzyme against proteolytic degradation and functional abnormalities; the possibility to compensate the deficiency by an overexpression of the corresponding isoenzyme or by the use of an alternative metabolic pathway. Difficulties in estimating the quantitative degree of disorder in severe cases are due to the fact that these populations contain many reticulocytes, which generally have higher enzyme activities and concentrations of intermediates than erythrocytes. An alternative approach to predict metabolic changes is the analysis by mathematical modeling. Mathematical modeling of the main metabolic pathways of human erythrocytes has reached an advanced level (Rapoport et al., 1976; Holzhütter et al., 1985; Schuster et al., 1988). Models have been successfully employed to describe stationary and time-dependent metabolic states of the cell under normal conditions as well as in the presence of enzyme deficiencies. Figure 5 shows computational results of erythrocyte enzyme deficiencies. This analysis is based on the comprehensive mathematical model of the energy and redox metabolism for human erythrocyte presented in Fig. 6. Stationary states of the cell metabolism have been calculated by varying the activity of each of the participating enzymes by several orders of magnitude. To predict consequences of enzyme deficiencies a performance function has been introduced (Schuster and Holzhütter, 1995). It takes into account the homeostasis of three essential metabolic variables: the energetic state (ATP), the reductive capacity (reduced glutathione) and the osmotic state. From the data given in Fig. 5 one can conclude that generally the metabolic impairment resulting in deficiencies occurs earlier for enzymes with high control coefficients than for those catalyzing equilibrium reactions. On the other hand the flux curves of latter enzymes decrease more steeply below a critica

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.

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)

Yeast genes encoding enzymes of the glycolytic pathway are highly expressed due to transcriptional control elements in their promoters. We provide data on such elements in the 5'-noncoding sequences of the Saccharomyces cerevisiae GPM1 gene, encoding phosphoglycerate mutase. Using fusions to the lacZ reporter gene, a detailed deletion analysis was performed. A palindromic sequence was shown to function as an upstream activation site (UAS) and two upstream repressing sites (URS1 and URS2) were located. Western and Northern blot analyses were used to substantiate the data obtained in enzymatic measurements. The regulatory sequences were shown to be functional in the heterologous CYC1 promoter. In addition, a promoter region was detected which mediated general glycolytic control by the GCR1 regulatory factor.

The glycolytic enzyme phosphoglycerate mutase (PGAM) is a dimer, and mature human skeletal muscle contains almost exclusively the MM form of the enzyme, PGAM-M. In 1981, we identified a patient with PGAM-M deficiency, and three additional patients have since been described. All presented with exercise intolerance, cramps, and myoglobinuria. We report two new patients with PGAM-M deficiency and describe the molecular lesions in five patients--four African-Americans and one Caucasian. Three patients were homozygous for an identical G-to-A transition converting an encoded Trp to an in-frame stop codon (codon 78). A fourth patient was heterozygous for this mutation and also carried an A-to-C mutation converting Glu to Ala (codon 89). The fifth patient, the only Caucasian, was homozygous for a different point mutation, a C-to-T mutation, converting Arg to Trp (codon 90).

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 Créteil I) and the other bearing a frameshift mutation (mutation BPGM Créteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Créteil I mutation.

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.

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.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.

Phosphoglycerate mutase consists of two kinds of different subunits, M and B. We previously sequenced a rat cDNA encoding the type-M subunit. Here, we report the sequence of the type-B subunit-encoding cDNA. This cDNA has 1754 bp and contains a long 3'-untranslated region of 897 bp.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.

A cDNA encoding the nonmuscle-specific (type B) subunit of phosphoglycerate mutase (PGAM-B) was isolated and characterized. A cDNA probe, synthesized by the polymerase chain reaction (PCR) from rat liver cell mRNA using mixed primers specific to the amino acid sequence of human PGAM-B, was used to screen a rat liver cell cDNA library. The identity of the cDNA was confirmed by amino acid sequence data for 24 peptides obtained by digesting the purified protein with three different endopeptidases. The coding region encoded a polypeptide composed of 253 amino acid (plus the initiator Met). RNA blot analysis showed a single mRNA species of 1.7 kilobases in rat liver cell. The deduced amino acid sequence of rat PGAM-B was identical to that of human PGAM-B except for only one substitution at position 251 near the carboxyl terminus (valine for the rat and alanine for the human).

In this study we report on the complete nucleotide sequence of the yeast phosphoglycerate mutase gene (GPM1) and its essential 5' and 3' non-coding regions. The transcriptional start points were determined by S1-mapping and sequencing of a cDNA clone. Several sequences identified as important for transcriptional regulation in yeast promoters are present upstream of the transcription start point. 3' to the coding region we sequenced a composite repetitive element which, apparently, originated from a recombination between a delta- and a tau-element. Finally, we mapped the GPM1 gene 13 cM distal to fas1 on chromosome XI.

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.

Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder that has been mapped to 7p13. We have investigated two patients with GCPS and a cytogenetically visible microdeletion of the short arm of chromosome 7 with gene probes that have been assigned close to the proposed Greig locus. Deletion breakpoints were determined from high-resolution G- and R-banded chromosomes. In patient BC with a de novo deletion (7p12.3-7p14.2) we have found a loss of the genomic region containing the T-cell receptor gamma (TCRG) gene cluster, whereas the other patient IR with a deletion (7p11.2-7p13) due to a de novo translocation was apparently normal for this region. Gene dosage analysis revealed a loss of the phosphoglycerate mutase muscular form (PGAM2) gene locus in both patients. Hox 1.4 and interferon-beta 2 (IFNB2) showed a normal gene dosage. Our investigations revealed the following ordering and assignments of the studied genes: PGAM2 and GCPS in 7p12.3-13; TCRG in the distal part of 7p13-7p14.2; Hox 1.4 and IFNB2 distal to 7p14.2. Our results suggest a location of the TCRG gene more proximal than that reported previously. Furthermore, we were able to exclude the Hox 1.4 gene from involvement in the pathogenesis of GCPS.

Poly(A)+ mRNA was isolated from rat satellite cell cultures and analyzed by Northern blot analyses for mRNA content of phosphoglycerate mutase (PGAM) isozymes. In non-differentiating satellite cells only PGAM-B mRNA was detected, but when cells were differentiated into myotubes, which undergo spontaneous contraction, mRNA for PGAM-M muscle-specific isozyme was also detected. This finding is in perfect concordance with the transition of PGAM isozymes encountered in the same cell cultures, and strongly supports a transcriptional control of PGAM expression throughout myogenesis independently of nerve influence.

The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.

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.

A 2.3-kb-long probe derived from the 5' flanking region, the first exon and part of the first intron of the human muscle-specific phosphoglycerate mutase gene (PGAM-M) (EC 5.4.2.1) was used to map the gene by "in situ" chromosomal hybridization. The structural gene for PGAM-M was assigned to chromosome 7p12-7p13; a single hybridization peak indicated that there is a single gene for this isozyme of PGAM, and confirmed results obtained by Southern blot hybridization.

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.

A 1.1-kb cDNA clone for human 2,3-bisphosphoglycerate mutase (BPGM) (EC 2.7.5.4) was used to map the structural gene to metaphase chromosomes. In situ hybridization experiments localized the human BPGM gene to chromosome 7 and, more precisely, to region 7q34----7q22.

The reaction mechanism of rat hepatic fructose-2,6-bisphosphatase involves the formation of a phosphohistidine intermediate. In order to determine the sequence around the active site histidine, the enzyme was incubated with [2-32P]fructose 2,6-bisphosphate, denatured, and treated with trypsin or endoproteinase Lys-C. The resultant labeled 32P-phosphopeptides were purified by gel filtration, anion exchange chromatography, and reverse phase high pressure liquid chromatography. The sequence of the tryptic peptide was determined to be HGESELNLR, while the partial sequence of the endoproteinase Lys-C peptide was IFDVGTRYMVNRVQDHVQSRTAYYLMNIHVTPRSIYLRHGESEL. The active site sequence was compared with the active site sequence of other enzymes that catalyze phospho group transfer via a phosphohistidine intermediate. Active site sequences of phosphoglycerate mutase and bisphosphoglycerate synthase were highly homologous with the active site of fructose-2,6-bisphosphatase implying a structural similarity and a common evolutionary origin.

The human 2,3-bisphosphoglycerate mutase gene was isolated from genomic libraries and analyzed by Southern blots and DNA sequencing. The transcription initiation site was localized by primer extension as well as by S1 protection of the mRNA. The gene extends over 22 kilobase pairs; it is composed of two introns (8.8 and 11.5 kilobase pairs long) and three exons (84, 662, and 965 base pairs long). The second exon correlates with a functional subdomain of the protein, as shown by comparison with the yeast phosphoglycerate mutase structure. The sequence TAGAAAA was found 30 bases upstream from the transcription initiation site and could be analogous to the TATA box. A sequence homologous to the CCAAT box was found twice, at positions -75 and -178. There is no GC-rich sequence or GC box in the 5'-flanking region of the gene. Northern blot analysis indicates that the 2,3-bisphosphoglycerate mutase mRNA is detected mainly in erythroid tissues and cell lines, although it is also present in low amounts in a nonerythroid tissue. A comparison of the 5'-upstream sequences with other promoters active only in erythroid cells did not reveal any common signal that could be responsible for the "erythroid promoter."

The Eskimos of St. Lawrence Island have been typed for genetic variation at 44 discrete genetic loci. Three private polymorphisms, at the 2,3-diphosphoglycerate mutase, peptidase B, and purine nucleoside phosphorylase loci, have been observed, which may be useful in future studies of genetic relationships between Eskimos and other circumpolar populations. Genetic distance analysis reveals a close relationship between the St. Lawrence Island Eskimos and other Eskimo populations and that the Eskimo populations form a distinct cluster from Amerindian populations. The St. Lawrence Island Eskimos appear to be more similar to Asiatic Eskimos than to other groups. Caucasian admixture in this population is estimated to be between 2 and 7%.

Phosphoglycerate mutase (GPM) functions reversibly in the glycolytic pathway. Mutations altering the reversibility of GPM have been obtained in the yeast, Saccharomyces cerevisiae. While wild-type cells grow on glycolytic (glucose) or gluconeogenic (ethanol) substrates, cells with altered GPMs fall into three categories based on their phenotypes 1) glucose- ethanol-, 2) glucose+ ethanol-, and 3) glucose- ethanol+. Cells with the first two phenotypes possessed GPMs that functioned irreversibly in the glycolytic direction. Cells that were glucose- ethanol+ possessed an enzyme that functioned reversibly. All of the altered GPMs had maximal velocities that were less than 3% of the wild-type level. The properties of the altered GPMs studied here provide a rationale for the occurrence in the glycolytic pathway of several glycolytic enzymes such as GPM, which function at high velocities in relation to the much smaller metabolic flux that they support. The altered GPMs were purified and estimates of their kinetic constants obtained. Free energy profiles were drawn for catalysis by the wild type and a mutant GPM that functioned irreversibly. The mutant enzyme was very inefficient. It was shown that an enzyme that functions irreversibly at a reaction with a Keq value close to 1 would necessarily be inefficient while it could evolve to be efficient when catalyzing a reaction that has a Keq value much greater than 1. In the glycolytic path this could be the reason for the characteristic presence of enzymes that function irreversibly at reactions with large Keq values.

We previously reported the isolation of a full-length cDNA specifying the muscle-specific isozyme of human phosphoglycerate mutase (PGAM-M). We now report the isolation of a full-length cDNA specifying the non-muscle-specific, or brain (B), isozyme of human PGAM (PGAM-B). The PGAM-B cDNA encodes a deduced protein 254 amino acids long, 79% identical to PGAM-M, and contains a 913-nucleotide 3'-untranslated region, as compared to the unusually short 37-nucleotide 3'-untranslated region of PGAM-M. Northern analysis demonstrates the non-muscle-specific nature of PGAM-B transcription, while genomic Southern analysis implies the presence of a large PGAM family in the human genome. Most of the PGAM-hybridizing sequences in both the human and mouse genomes seem to be related to the B-isozyme gene; many members of the PGAM-B gene family in humans are apparently processed genes. These results agree with the evolutionary analysis, which indicates that the PGAM-B gene is the progenitor of the PGAM-M gene.

We examined developmental changes in the relative activities of three different isozyme systems: aldolase, enolase and phosphoglycerate mutase, in tissues of fetal mice with trisomy 16 and of fetal euploid littermates. We wanted to determine whether morphological abnormalities such as reduced weight and size, which are generally observed in murine trisomy, are reflected at the molecular level. Following electrophoretic separation and subsequent measurement of relative activities of enolase isozymes in brain and phosphoglycerate mutase isozymes in heart, we found no significant differences between trisomy 16 fetuses and their euploid littermates. Synthesis of liver-specific aldolase was, however, delayed in trisomy 16 fetuses.

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.

The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined. The gene specifies a protein of 246 amino acids, and contains no introns. The sequence shows a strong codon bias. The upstream untranslated portion of the gene contains a CT-rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence.

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.

2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. The synthase activity of both enzymes shows a nonhyperbolic pattern which fits to a second degree polynomial. The Km, Ki and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.