Muscle Phosphoglycerate Mutase Research Articles

High-Resolution Crystal Structure of Muscle Phosphoglycerate Mutase Provides Insight into Its Nuclear Import and Role.

Phosphoglycerate mutase (PGAM) is a glycolytic enzyme converting 3-phosphoglycerate to 2-phosphoglycerate, which in mammalian cells is expressed in two isoforms: brain (PGAM1) and muscle (PGAM2). Recently, it was shown that besides its enzymatic function, PGAM2 can be imported to the cell nucleus where it co-localizes with the nucleoli. It was suggested that it functions there to stabilize the nucleolar structure, maintain mRNA expression, and assist in the assembly of new pre-ribosomal subunits. However, the precise mechanism by which the protein translocates to the nucleus is unknown. In this study, we present the first crystal structure of PGAM2, identify the residues involved in the nuclear localization of the protein and propose that PGAM contains a "quaternary nuclear localization sequence (NLS)", i.e., one that consists of residues from different protein chains. Additionally, we identify potential interaction partners for PGAM2 in the nucleoli and demonstrate that 14-3-3ζ/δ is indeed an interaction partner of PGAM2 in the nucleus. We also present evidence that the insulin/IGF1-PI3K-Akt-mTOR signaling pathway is responsible for the nuclear localization of PGAM2.

CARM1/PRMT4 is necessary for the glycogen gene expression programme in skeletal muscle cells

CARM1 (co-activator-associated arginine methyltransferase 1)/PRMT4 (protein arginine methyltransferase 4), functions as a co-activator for transcription factors that are regulators of muscle fibre type and oxidative metabolism, including PGC (peroxisome-proliferator-activated receptor γ co-activator)-1α and MEF2 (myocyte enhancer factor 2). We observed significantly higher Prmt4 mRNA expression in comparison with Prmt1-Prmt6 mRNA expression in mouse muscle (in vitro and in vivo). Transfection of Prmt4 siRNA (small interfering RNA) into mouse skeletal muscle C2C12 cells attenuated PRMT4 mRNA and protein expression. We subsequently performed additional qPCR (quantitative PCR) analysis (in the context of metabolism) to examine the effect of Prmt4 siRNA expression on >200 critical genes that control (and are involved in) lipid, glucose and energy homoeostasis, and circadian rhythm. This analysis revealed a strikingly specific metabolic expression footprint, and revealed that PRMT4 is necessary for the expression of genes involved in glycogen metabolism in skeletal muscle cells. Prmt4 siRNA expression selectively suppressed the mRNAs encoding Gys1 (glycogen synthase 1), Pgam2 (muscle phosphoglycerate mutase 2) and Pygm (muscle glycogen phosphorylase). Significantly, PGAM, PYGM and GYS1 deficiency in humans causes glycogen storage diseases type X, type V/McArdle's disease and type 0 respectively. Attenuation of PRMT4 was also associated with decreased expression of the mRNAs encoding AMPK (AMP-activated protein kinase) α2/γ3 (Prkaa2 and Prkag3) and p38 MAPK (mitogen-activated protein kinase), previously implicated in Wolff-Parkinson-White syndrome and Pompe Disease (glycogen storage disease type II). Furthermore, stable transfection of two PRMT4-site-specific (methyltransferase deficient) mutants (CARM1/PRMT4 VLD and CARM1E267Q) significantly repressed the expression of Gys1, Pgam2 and AMPKγ3. Finally, in concordance, we observed increased and decreased glycogen levels in PRMT4 (native)- and VLD (methylation deficient mutant)-transfected skeletal muscle cells respectively. This demonstrated that PRMT4 expression and the associated methyltransferase activity is necessary for the gene expression programme involved in glycogen metabolism and human glycogen storage diseases.

Phosphoglycerate mutase in mammalian striated muscles: Subcellular localization and binding partners

Contrary to previously published data, we have found that in mammalian skeletal muscles, phosphoglycerate mutase (PGM) is organized in a regular, striated fashion within the sarcomere. In the absence of the enzyme effectors, PGM localizes mainly at the M-line, but under conditions typical for contracting muscle, the enzyme accumulates within the I-band of the sarcomere. Searching for muscle PGM binding partners, we have found that PGM interacts with several enzymes of triose phosphate metabolism. It might suggest that PGM is a central structural element of the muscle glycolytic complex located within the isotropic region of the sarcomere. Structured summary MINT- 7034028: PGM (uniprotkb: P16290) physically interacts (MI: 0218) with lactate dehydrogenase B (uniprotkb: P42123), lactate dehydrogenase A (uniprotkb: P04642), G3PDH (uniprotkb: P04797), aldolase (uniprotkb: P05065), Creatine kinase (uniprotkb: P07335), phPhosphoglycerate kinase (uniprotkb: P16617) and Enolase (uniprotkb: P04764) by pull down (MI: 0096)

G.P.16.08. Two novel mutations associated with muscle phosphoglycerate mutase (PGAM) deficiency

G.P.16.08. Two novel mutations associated with muscle phosphoglycerate mutase (PGAM) deficiency

Effect of Fuels on Exercise Capacity in Muscle Phosphoglycerate Mutase Deficiency

Muscle phosphoglycerate mutase deficiency (PGAMD) is a rare, recessively inherited metabolic myopathy that affects one of the last steps of glycolysis. Clinically, PGAMD resembles muscle phosphorylase deficiency (McArdle disease) and phosphofructokinase deficiency (PFKD). However, it is unknown whether PGAMD is associated with a second-wind phenomenon during exercise, as in McArdle disease, and whether patients with PGAMD, like patients with PFKD and McArdle disease, benefit from supplementation with fuels that bypass the metabolic block. To investigate whether fuels that bypass the metabolic block can improve exercise capacity or whether exercise capacity improves during sustained exercise. Single-blind, placebo-controlled investigation of the effects of glucose, lactate, and intralipid on work capacity in patients with PGAMD. National University Hospital, University of Copenhagen, and Neuromuscular Center, Institute for Exercise and Environmental Medicine. Patients Two unrelated men (21 and 26 years old) with PGAMD who since their teens had experienced muscle cramps, muscle pain, and episodes of myoglobinuria provoked by brief vigorous exercise, 4 patients with McArdle disease (mean +/- SD age, 32 +/- 5 years) with 0% residual phosphorylase activity in muscle, and 6 healthy, untrained male volunteers (mean +/- SD age, 23 +/- 1 years) were studied. Using constant and variable workload protocols on a cycle ergometer, it was investigated whether a spontaneous second wind occurs during exercise in patients with PGAMD, and using a constant workload protocol followed by an incremental load to exhaustion, it was tested whether infusion of lactate, glucose, or intralipid alters the exercise tolerance in PGAMD. Whether a second wind occurs during exercise and whether fuels that bypass the metabolic block can improve exercise and oxidative capacity. In contrast to patients with McArdle disease, with whom they share many clinical features, in patients with PGAMD, cycle exercise and oxidative capacity are virtually normal, a second wind does not occur, and lipid and lactate supplements do not improve exercise capacity. Although the clinical manifestations of PGAMD mimic McArdle disease with respect to the presence of exertional muscle cramps, rhabdomyolysis, and myoglobinuria, this study shows that cycle exercise responses are strikingly different.

Inactivation of phosphoglycerate mutase and creatine kinase isoenzymes in human serum

Aims/Background: Total phosphoglycerate mutase (PGM) activity in serum has been shown to be increased in acute myocardial infarction with the same time course as creatine kinase (CK) activity. However, the...

Effects of Thyroid Hormone on mRNAs of Phosphoglycerate Mutase Subunits in Rat Muscle during Development

Background: We previously showed that triiodothyronine (T3) stimulates muscle phosphoglycerate mutase (PGAM) activity and isozyme transition in rat skeletal and cardiac muscles. Methods: The effects of T3 on PGAM types B and M subunit expression in rat muscle during development are reported. Results: T3 administration during the first 21 days of rat life more than doubles type M PGAM mRNA levels, but produces minor effects on type B PGAM mRNA levels. The antihormone propylthiouracil (PTU) slightly decreases both type B and M mRNA levels, but this decrease is not statistically significant. Conclusion: Thyroid hormone influences PGAM mRNA isozyme levels differently and increases type M mRNA.

Muscle phosphoglycerate mutase deficiency with tubular aggregates: Effect of dantrolene

Muscle phosphoglycerate mutase deficiency with tubular aggregates: Effect of dantrolene

Muscle phosphoglycerate mutase deficiency with tubular aggregates: Effect of dantrolene

A patient with muscle phosphoglycerate mutase deficiency (PGAMD) and exercise-induced muscle cramps had tubular aggregates in muscle and increased muscle Ca2+-adenosine triphosphatase and calcium content. Two ischemic forearm exercise tests induced contractures in the patient. On dantrolene treatment, the patient became asymptomatic, and the ischemic test was performed without contracture. These findings suggest that cramps in muscle PGAMD are caused by a high calcium release from the sarcoplasmic reticulum relative to calcium re-uptake capacity.

Molecular genetic studies in muscle phosphoglycerate mutase (PGAM-M) deficiency.

Phosphoglycerate mutase (PGAM; EC 2.7.5.3) catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic pathway. Hereditary muscle PGAM deficiency has been identified in 9 patients with myopathy. All patients had exercise intolerance and 6 had myoglobinuria. Seven of the 9 patients were African-Americans: 5 of them were homozygous for a nonsense mutation, TGG(Trp) to TAG at codon 78; 1 was a compound heterozygote for the nonsense mutation and a missense mutation, GAG(Glu) to GCG(Ala) at codon 89; and 1 could not be tested. The only 2 Caucasian patients, a brother and sister, were homozygous for a different missense mutation, CGG(Arg) to TGG(Trp) at codon 90. Despite the small number of patients identified, these findings indicate that there is a common mutation in African-Americans while there may be molecular genetic heterogeneity in other ethnic groups.

Sequence of rat skeletal muscle phosphoglycerate mutase cDNA

A cDNA clone coding rat skeletal muscle phosphoglycerate mutase was isolated from a rat muscle lambda gt10 cDNA library and its sequence was determined. The deduced protein possesses 252 amino acids and is 94% homologous with respect to human muscle phosphoglycerate mutase. No amino acids changes occur at the active site and structural predictions suggest strong conformational homologies with other enzymes of the mutase family.

Purification of human muscle phosphoglycerate mutase by fast protein liquid chromatography based on hydrophobic interactions

Purification of human muscle phosphoglycerate mutase by fast protein liquid chromatography based on hydrophobic interactions

2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate.

The binding of inorganic vanadate (Vi) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [Vi]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 X 10(-6) M. The relevance of this result to other studies which have shown that the Vi-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [Vi] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 microM Vi has little effect on the phosphatase activity. However, in the presence of 25 microM Vi, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed.

The catalytic bimodality of mammalian phosphoglycerate mutase.

Rabbit muscle phosphoglycerate mutase, presumed to manifest an absolute cofactor requirement for activity, has been found to express catalysis (3 +/- 1% of optimum) in the absence of added D-glycerate-2,3-P2. Isotope experiments indicate that this catalysis proceeds through a binary phosphoryl enzyme-glycerate intermediate which dissociates into free enzyme and monophosphoglycerate. 32P-Labeled phosphoglycerate mutase is formed by reaction with either D-32P-glycerate-3-P or D-U32P-glycerate-2,3-P2. In each case, the acid lability and alkali stability of the covalent adduct, phosphoenzyme, is consistent with a phosphohistidyl residue having been formed within the active site. D-[U-14C]Glycerate reacts with phosphoenzyme to generate D-[U-14C]monophosphoglycerate which, in turn, can react further with phosphoenzyme to yield D-[U-14C]glycerate-2,3-P2. The pH profile for the cofactor-independent activity exhibits an optimum at 6.0 as opposed to 7.0 when D-glycerate-2,3-P2 is present in the reaction medium. Bisubstrate kinetics (pH 7.0, 23 degrees C) with D-glycerate-3-P concentration as the variable, yields a family of reciprocal plots which is in accord with a modified ping-pong mechanism when D-glycerate-2,3-P2 concentrations are greater than 10(-1) Km (where Km = 0.33 microM). Progressively diminishing concentrations (much less than Km) of D-glycerate-2,3-P2 produce curvilinear reciprocal plots that approach linearity as a limit in accordance with single substrate kinetics.

Inactivation of rabbit muscle phosphoglycerate mutase by limited proteolysis with thermolysin.

Rabbit muscle phosphoglycerate mutase is inactivated by proteolysis with thermolysin. Inactivation is correlated with the breakage of one (or a few) bond(s) near one end of the polypeptide chain. There is no change in the overall conformation, quaternary structure or binding to Cibacron Blue on proteolysis. The possible analogy with the existence of a flexible tail in the yeast enzyme is discussed.

Metabolism of glycerate-2,3-P 2—VI. Lysyl-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with pyridoxal-5′-phosphate produces the concurrent loss of the three activites of the enzyme: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. 3. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P 2. 4. 4. Substrate (glycerate-3-P) protects against pyridoxal-5′-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P 2) does not prevent inactivation. 5. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favour their location at the same active site. 6. 6. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.

Metabolism of glycerate-2,3-P 2—V. Histidine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Both treatment with diethylpyrocarbonate and photo-oxidation with rose bengal produces the loss of the three enzymatic activities of rabbit muscle phosphoglycerate mutase: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. The synthase and the phosphatase activities are less affected than the mutase activity. 3. 3. Glycerate-2,3-P 2 and glycerate-3-P protect against diethylpyrocarbonate inactivation, but do not protect against inactivation produced by photo-oxidation. 4. 4. Hydroxylamine reactivates the diethylpyrocarbonate-treated enzyme. 5. 5. Chemical modification of phosphoglycerate mutase markedly reduces its ability to form the functionally active phosphoenzyme. 6. 6. These results provide evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase. In addition, they support the existence of two separate binding sites for monophosphoglycerates and for bisphosphoglycerates.

Metabolism of glycerate-2,3-P 2—III. Arginine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Treatment of rabbit muscle phosphoglycerate mutase with diketones (2,3-butanedione and 1,2-cyclohexanedione) and with glyoxal derivatives (methylglyoxal and phenylglyoxal) produces the loss of the three activities of the enzyme: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. Hydroxylamine reactivates all the activities of the modified enzyme. 3. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P 2. 4. 4. Both substrate and cofactor protect against inactivation. 5. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favor their location at the same active site. 6. 6. In addition, they suggest that arginine is involved in the binding of the cofactor to the enzyme.

Phylogeny and ontogeny of the phosphoglycerate mutases—III. Inactivation of rabbit muscle phosphoglycerate mutase (type M isozyme) by the sulfhydryl group reagents

1. 1. The three phosphoglycerate mutase isozymes from mammals (types M, B and MB isozymes) differ in their sensitivity to the —SH group reagents. 2. 2. Rabbit muscle phosphoglycerate mutase (type M isozyme) is reversibly inactivated by tetrathionate, p-chloromercuribenzoate and Hg 2+. 3. 3. Titration with p-chloromercuribenzoate shows the existence of two sulfhydryl groups per enzyme subunit, the modification of which produces a progressive decline in enzyme activity. 4. 4. The apparent K m values for substrate and cofactor are not affected by tetrathionate treatment. 5. 5. Phosphoglycerate mutase inactivated by tetrathionate and by p-chloromercuribenzoate is unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P 2, and is not protected by the cofactor against heating. 6. 6. Glycerate-2,3-P 2 protects against tetrathionate treatment, but fails to protect against Hg 2+ and p-chloromercuribenzoate inactivation.

Phylogeny and ontogeny of the phosphoglycerate mutases—v. inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. 1. The three isozymes of glycerate-2,3-P 2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 2. 2. Inactivation of type M isozyme purified from rabbit muscle was complete when two histidine residues per enzyme subunit were carboethoxylated. Hydroxylamine removed the carboethoxy groups, with partial recovery of the enzymatic activity. The cofactor protected the enzyme against inactivation. 3. 3. The inactivation of rabbit muscle phosphoglycerate mutase by photooxidation with methylene blue and rose bengal was sharply pH dependent. The pH profile of enzyme inactivation followed the titration curve of histidine, suggesting that this amino acid was critical for enzyme activity. Glycerate-2,3-P 2 did not protect phosphoglycerate mutase against photoinactivation.