Metabolism of glycerate-2,3-P2--IV. Effect of Hg2+ on the enzymes involved in the metabolism of glycerate-2,3-P2 in pig skeletal muscle

Type M phosphoglycerate mutase and skeletal muscle bisphosphoglycerate synthase-phosphatase from pig are similarly affected by Hg2+. Both enzymes lose the phosphoglycerate mutase and the glycerate-2,3-P2 synthase activities, and increase the glycerate-2,3-P2 phosphatase activity upon Hg2+-treatment. In contrast, bisphosphoglycerate phosphatase from pig skeletal muscle is inactivated by Hg2+. These results confirm the similarity between phosphoglycerate mutase and bisphosphoglycerate synthase-phosphatase. In addition they support the existence of separate binding sites for monophosphoglycerates and for bisphosphoglycerates at the phosphoglycerate mutase active site.     

Metabolism of glycerate-2,3-P2--VII. Enzymes involved in the metabolism of glycerate-2,3-P2 in cat tissues

The levels of the enzymes involved in the metabolism of glycerate-2,3-P2 (phosphoglycerate mutase, bisphosphoglycerate synthase-phosphatase and bisphosphoglycerate phosphatase) in cat and in pig tissues are different. The main difference is the low level of bisphosphoglycerate synthase-phosphatase in cat tissues. As a consequence, in contrast with pig erythrocytes, in cat erythrocytes, both the synthesis and the breakdown of glycerate-2,3-P2 are mainly controlled by phosphoglycerate mutase.     

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).     

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.     

Kinetic properties and essential amino acids of the 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle

Histidine, arginine and lysine residues are essential for the multifunctional 2,3-bisphosphoglycerate synthase-phosphatase purified from pig skeletal muscle. The synthase, phosphatase and phosphoglycerate mutase activities of the enzyme are concurrently lost upon treatment with diethylpyrocarbonate, phenylglyoxal and trinitrobenzenesulfonate. The phosphatase activity shows hyperbolic kinetics. In contrast, the synthase activity shows a nonhyperbolic pattern which fits to a second-degree polynomial. The Km values for glycerate 1,3-P2, glycerate 3-P and glycerate 2,3-P2 are similar to those of the enzyme from mammalian erythrocytes.     

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.     

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).     

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.     

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.     

Evidence for structural homology between human red cell phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase.

Previous reports have suggested the possibility of extensive structural homology between human erythrocyte bisphosphoglycerate synthase (glycerate-1,3-P2 leads to glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P in equilibrium glycerate-2-P). This study lends credence to that conjecture through comparative physicochemical investigations involving peptide mapping, circular dichroism, and immunological techniques. The data indicate that despite differences in function, both enzymes apparently manifest a high degree of similarity in primary, secondary, and tertiary structure. Mapping data also indicate that each protein is comprised of two apparently identical subunits.     

Purification and characterization of phosphoglycerate mutase isozymes from pig heart

The three isozymes of phosphoglycerate mutase from pig heart have been purified to homogeneity. The isozymes have a molecular weight of 57000 as determined by gel-filtration chromatography. Discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate yields a single band with a molecular weight of 29000, indicating that the isozymes are dimers composed of subunits of similar mass. Hybridization experiments show that the three isozymes result from homodimeric and heterodimeric combinations of two different subunits. The two types of subunit differ in their heat lability and in the presence of -SH groups essential for enzymatic activity. No remarkable differences exist in the kinetic constants of the purified isozymes. The kinetic pattern is consistent with a 'ping-pong' mechanism. The homogeneous preparations of the three isozymes show intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity which can be stimulated by glycolate-2-P.     

Active site phosphohistidine peptides from red cell bisphosphoglycerate synthase and yeast phosphoglycerate mutase.

Bisphosphoglycerate synthase (glycerate-1,3-P2 yields glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P formed from glycerate-2-P) are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes (Rose, Z. B., and Dube, S. (1976) J. Biol. Chem. 251, 4817--4822). We have phosphorylated bisphosphoglycerate synthase from horse red blood cells with [U-32P]glycerate-2,3-P2, digested with trypsin, and purified the phosphopeptide. The amino acid sequence of the phosphohistidine peptide has been determined to be: His-Gly-Gln-Gly-Ala-Trp-Asn-Lys. In like manner, a phosphohistidyl peptide has now been purified from yeast phosphoglycerate mutase, for which the amino acid sequence is known (Winn, S. I., Watson, H. C., Fothergill, L. A., and Harkins, R. N. (1977) Biochem. Soc. Trans. 5, 657-659). The amino acid composition of the phosphopeptide indicates that histidine-8 was phosphorylated. The sequence of this peptide is closely homologous with the active site peptide from bisphosphoglycerate synthase. In yeast phosphoglycerate mutase, the denatured phosphoenzyme hydrolyzes with a single rate constant of 2.02 X 10(-4) s-1 at pH 3, 45 degrees C. The relevance of these observations to the enzymatic mechanism is discussed.     

Phylogeny and ontogeny of the phosphoglycerate mutases.--V. Inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. The three isozymes of glycerate-2,3-P2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 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. 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-P2 did not protect phosphoglycerate mutase against photoinactivation.     

Synthesis of 2,3-bisphosphoglycerate synthase in erythroid cells

Antiserum prepared from a rabbit which was immunized with human erythrocyte glycerate-2,3-P2 synthase was found to react with glycerate-2,3-P2 synthase in rabbit erythroid cells. By using this antiserum, it was proved that the specific activity of this enzyme was unchanged during the development of the rabbit erythroid cells. This leads us to conclude that the increased activity of glycerate-2,3-P2 synthase in developing erythroid cells (Narita, H., Ikura, K., Yanagawa, S., Sasaki, R., Chiba, H., Saimyoji, H., and Kumagai, N. (1980) J. Biol. Chem. 255, 5230-5235) is due to the accumulation of enzyme protein. There is at least a 16-fold increase in the level of this protein during development from bone marrow erythroid cells to erythrocytes. The synthesis of glycerate-2,3-P2 synthase was shown to occur in rabbit reticulocytes and bone marrow erythroid cells. These cells were incubated for protein synthesis and the protein synthesized was precipitated with the anti-glycerate-2,3-P2 synthase antiserum and separated on sodium dodecyl sulfate-polyacrylamide gels. The immunoprecipitated product was shown to produce fragments of the same molecular weight after digestion with V8 protease as did the pure glycerate-2,3-P2 synthase. The proportion of glycerate-2,3-P2 synthase synthesis in reticulocytes (0.04% of total protein synthesis) was comparable to the level of this protein in the cells (0.07% of the total protein).     

Changes in the enzymes related to 2,3-bisphosphoglycerate metabolism in developing erythroid cells

Measurements of glycerate-2,3-P2 and hemoglobin in the developing erythroid cells indicated that the glycerate-2,3-P2 level rose during erythroid differentiation in a linear relationship to the hemoglobin level, suggesting the presence of regulation to accumulate both substances synchronously. The accumulation of glycerate-2,3-P2 was found to be primarily attributable to the increase in glycerate-2,3-P2 synthase activity. The activities of phosphofructokinase and pyruvate kinase changed so as to be favourable for glycerate-2,3-P2 accumulation. The increase in glycerate-2,3-P2 synthase activity was shown to be caused by an increase in the enzyme protein. Synthesis of glycerate-2,3-P2 synthase protein was proved in bone marrow erythroid cells and in reticulocytes.     

The purification and kinetic properties of biophosphoglycerate synthase from horse red blood cells.

Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P₂ or glycerate-2,3-P₂ to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P₂ → glycerate-2,3-P₂), k_cat 12.5 s^?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P₂ → glycerate-P + P_i), k_cat 2.6 s^?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), k_cat 1.7 s^?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell.     

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

1. The three phosphoglycerate mutase isozymes from mammals (types M, B and MB isozymes) differ in their sensitivity to the - SH group reagents. 2. Rabbit muscle phosphoglycerate mutase (type M isozyme) is reversibly inactivated by tetrathionate, rho-chloromercuribenzoate and Hg2+. 3. Titration with rho-chloromercuribenzoate shows the existence of two sulfhydryl groups per enzyme subunit, the modification of which produces a progressive decline in enzyme activity. 4. The apparent Km values for substrate and cofactor are not affected by tetrathionate treatment. 5. Phosphoglycerate mutase inactivated by tetrathionate and by rho-chloromercuribenzoate is unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P2, and is not protected by the cofactor against heating. 6. Glycerate-2,3-P2 protects against tetrathionate treatment, but fails to protect against Hg2+ and rho-chloromercuribenzoate inactivation.     

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.     

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.     

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

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