Vanadate affects glucose metabolism of human erythrocytes

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons and G. Pons (1982) Biochim. Biophys. Acta705, 238–242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.     

Phosphoglycerate mutase from wheat germ: studies with 18O-labeled substrate, investigations of the phosphatase and phosphoryl transfer activities, and evidence for a phosphoryl-enzyme intermediate.

From studies using unlabeled phospho-D-glycerate in solutions enriched in H2(18)O, and from experiments involving [18O]phospho-D-glycerate, it is shown that the intramolecular isomerization of 2- and 3-phospho-D-glycerate that is catalyzed by the phosphoglycerate mutase from wheat germ does not involve an intermediate 2,3-cyclic phosphate. It is also shown that phosphoglycerate mutase catalyzes the hydrolysis of the substrate analogues 2-phosphoglycolate, 2-phospho-D-lactate, 3-phosphohydroxypropionate, phosphoenolpyruvate, and phosphohydroxypyruvate. The substrates 3- and 2-phospho-D-glycerate are not hydrolyzed, nor are 2,3-bisphospho-D-glycerate, 2-phospho-L-lactate, 3-phospho-L-glycerate, or sn-glycerol 3-phosphate. Although no exchange of D-[14C]glycerate into phospho-D-glycerate can be detected, the enzyme catalyzes the transfer of the phosphoryl group from "unnatural" donors such as 2-phosphoglycolate, to the "natural" acceptor, D-glycerate. It is concluded that the intramolecular phosphoryl transfer catalyzed by the wheat germ phosphoglycerate mutase follows a pathway involving a phosphoryl-enzyme intermediate.     

Functional homology of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, phosphoglycerate mutase, and 2,3-bisphosphoglycerate mutase

The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast phosphoglycerate mutase and human red blood cell 2,3-bisphosphoglycerate mutase including very similar active site sequences with a histidyl residue being involved in phospho group transfer. The liver bifunctional enzyme was found to catalyze the hydrolysis of glycerate 1,3-bisphosphate to glycerate 3-phosphate and inorganic phosphate. The Km for glycerate 1,3-bisphosphate was 320 microM and the Vmax was 11.5 milliunits/mg. Incubation of the rat liver enzyme with [1-32P]glycerate 1,3-bisphosphate resulted in the formation of a phosphoenzyme intermediate, and the labeled amino acid was identified as 3-phosphohistidine. Tryptic and endoproteinase Lys-C peptide maps of the 32P-phosphoenzyme labeled either with [2-32P]fructose 2,6-bisphosphate or [1-32P]glycerate 1,3-bisphosphate revealed that 32P-radioactivity was found in the same peptide, proving that the same histidyl group accepts phosphate from both substrates. Fructose 2,6-bisphosphate inhibited competitively the formation of phosphoenzyme from [1-32P]glycerate 1,3-bisphosphate. Effectors of fructose-2,6-bisphosphatase also inhibited phosphoenzyme formation. Substrates and products of phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase also modulated the activities of the bifunctional enzyme. These results demonstrate that, in addition to a structural homology, the bisphosphatase domain of the bifunctional enzyme has a functional similarity to phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase and support the concept of an evolutionary relationship between the three enzyme activities.     

Functional characterization of the enzymes with 2,3-bisphosphoglycerate phosphatase activity from pig skeletal muscle

In pig skeletal muscle exist four enzymes with 2,3-bisphosphoglycerate phosphatase activity. Two of them (forms I-A and I-C) are multi-functional enzymes which, in addition to the phosphatase activity, possess 2,3-bisphosphoglycerate synthase and phosphoglycerate mutase activities. The other two enzyme forms (II-A and II-B) only show the phosphatase activity. The four enzymes differ in substrate specificity. Form I-C is highly specific for glycerate 2,3-P2; form I-A also hydrolyzes the monophosphoglycerates and forms II-A and II-B are specific for phosphoester bonds adjacent to a C-1 carboxylic group. The enzymes possess similar Km, Kcat and optimum pH value, but they are differently inhibited by the reaction products. They are also differently affected by glycolate-2-P (their main activator) and by other modifiers. Probably form I-A, which corresponds to M-type phosphoglycerate mutase, is the main enzyme implicated in the breakdown of glycerate 2,3-P2 in pig muscle.     

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

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.     

Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle.

The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.     

2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Molecular cloning and nucleotide sequence of murine 2,3-bisphosphoglycerate mutase cDNA

Cloning and sequencing of a murine cDNA with the entire coding region of 2,3-bisphosphoglycerate mutase is reported, as a prerequisite for further expression studies of this erythroid specific enzyme in Friend mouse erythroleukemia cells. A comparison between species of the deduced amino acid sequences of these proteins shows 20 substitutions between mouse and human and 21 between mouse and rabbit: none of these substitutions are in positions assumed to be in the active site. Amino acid alignment with the other related enzymes, the phosphoglycerate mutases, in combination with crystallographic data from yeast phosphoglycerate mutase, gives some insight into the structure/function correlation for this protein family. Amino acid residues which are most likely critical for either 2,3-bisphosphoglycerate mutase or phosphoglycerate mutase function are pointed out. Concerning the phylogenetic analysis, phosphoglycerate mutases B and M from mammalians appear to have diverged with the yeast enzyme from a common ancestor, before the emergence of the 2,3-bisphosphoglycerate mutases.     

Hybrid forms of phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase-phosphatase

Purified phosphoglycerate mutase from pig skeletal muscle and 2,3-bisphosphoglycerate synthase-phosphatase from pig erythrocytes were hybridized “in vitro”. The hybrid showed a behaviour on electrophoresis and on ion-exchange chromatography similar to that of a naturally occurring enzyme with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities present in pig skeletal and heart muscle. Both the hybrid and the muscle enzyme possess similar activities ratio. From these and previous data it is suggested that the six enzymatic forms with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities detected in mammalian tissues (Carreras et al. 1981, Comp. Biochem. Physiol. 70B, 477–485) result from combination of three subunits (types M, B and E).     

Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle

Two enzymes which possess 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities have been purified from pig skeletal muscle. One of the enzymes corresponds to type M phosphoglycerate mutase. The other enzyme shows properties similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase present in mammalian erythrocytes. The erythrocyte and the muscle enzyme possess the same molecular (56 000) and subunit (27 000) weights. The synthase, phosphatase and mutase activity ratio is similar in both enzymes, and they are affected by the same inhibitor (glycerate 3-P) and activators (glycolate 2-P, pyrophosphate, sulfite and bisulfite).     

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.     

Metabolism of glycerate 2,3-P2--XI. Essential amino acids of pig phosphoglycerate mutase isozymes

Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. The reversion of the modifying reactions reactivates the three enzymatic activities. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast.     

Metabolism of glycerate 2,3-P2--XII. Characterization of the 2,3-bisphosphoglycerate synthase-phosphatase and of the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase from pig brain

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.     

PH-dependent changes of 2,3-bisphosphoglycerate in human red cells during transitional and steady states in vitro.

A systematic study of the pH-dependent changes in the range 6.6--7.4 of 2,3-bisphosphoglycerate and the adenine nucleotides was performed in the presence and absence of glucose during transitional and steady states. 1. The results indicatethat 2,3-gisphosphoglycerate phosphatase breaks down 2,3-bisphosphoglycerate nearly independent of pH at a rate of 480 mumol 2,3-bisphosphoglycerate x1 cells-1xh-1.2,3-Bisphosphoglycerate mutase is practically completely inhibited below pH value increases in long-term experiments with lower 2,3-bisphosphoglycerate levels. The formation of pyruvate corresponds to the breakdown of 2,3-bisphosphoglycerate afterconsumption of an unknown reducing substance.     

Increase of 2,3-bisphosphoglycerate synthase/phosphatase during maturation of reticulocytes with high 2,3-bisphosphoglycerate content

In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erytrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC 2.7.5.4/EC 3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.     

Crystal structure of human bisphosphoglycerate mutase

Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 A and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and R(free) of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98-117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed.     

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.     

Magnetic resonance studies of the interaction of divalent metal cations with 2,3-bisphosphoglycerate

The binding of Mg²⁺ to intracellular 2,3-bisphosphoglycerate in the human red blood cell is significant to the function of the cell. We have studied interactions of Mg²⁺ and Mn²⁺ with 2,3-bisphosphoglycerate by magnetic resonance spectroscopy. The results of this study reveal the presence of two independent divalent metal cation binding sites of similar affinity (K_D = 3.0 ± 0.5 mM) in the 2,3-bisphosphoglycerate molecule, one on each phosphoryl group, contrary to the assumption of one metal ion binding site made in the previous literature. Over the range of their intracellular concentrations, ATP and ADP, however, possess only one metal ion site in spite of the presence of multiple phosphoryl groups. These results are consistent with the chemistry of metal-chelation which requires the formation of 5- or 6-membered rings for the stability of chelate structures.     

磷酸甘油酸变位酶

磷酸甘油酸变位酶(phosphoglycerate mutase,PGM)是糖代谢过程中的关键酶,催化3-磷酸甘油酸和2-磷酸甘油酸之间的相互转换。根据催化反应中对辅因子2,3-二磷酸甘油酸的依赖关系分为两种类型:辅因子依赖型PGM(dPGM)和辅因子非依赖型PGM(iPGM)。本文对PGM的分类、结构及功能进行了详细介绍。     

Mechanism of phosphoacetylglucosamine mutase.

Kinetic studies of phosphoacetylglucosamine mutase (EC 2.7.5.2) for the following reactions: 1) Glc-1-P in equilibrium Glc-6-P and 2) GlcNAc-1-P in equilibrium GlcNAc-6-P have been conducted in the presence of Glc-1,6-P2 and GlcNAc-1,6-P2, respectively. In the first reaction, the initial velocity studies at various concentrations of one substrate showed a series of parallel lines in the Line-weaver-Burk plot when the concentrations of the other substrate were changed at several fixed levels. For both reactions, the initial velocity studies performed at fixed ratios of both substrates showed linear lines in the double reciprocal plot. The competitive substrate inhibition pattern was observed in the second reaction. A ping-pong mechanism is proposed for phosphoacetyl-glucosamine mutase. In addition, phosphoacetylglucosamine mutase can be phosphorylated by the addition of Glc-1-[32P]P probably via the reaction of Glc-1-[32P]P with the phosphoenzyme followed by the release of glucose-monophosphate leaving the 32P with the phosphoenzyme. The linkage between the phosphoryl residue and enzyme is stable in acid, but labile in alkali, suggesting phosphoserine (or phosphothreonine) as the phosphorylated amino acid. Biphasic heat denaturation curves suggest the existence of heat-stable and heat-labile forms of this enzyme.