Metabolism of glycerate-2,3-P2--II. Enzymes involved in the glycerate-2,3-P2 metabolism in chicken skeletal muscle

1. Four enzyme fractions which may be involved in the synthesis and breakdown of glycerate-2,3-P2 have been isolated from extracted skeletal muscle by gel-filtration and ion-exchange chromatography. 2. One of the fractions, corresponding to the glycerate-2,3-P2 dependent phosphoglycerate mutase, has been purified to homogeneity. In addition to the main enzymatic activity, it shows intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity stimulable by glycolate-2-P. Its synthase activity represents about 10% of the total synthase activity of the tissue, and its phosphatase activity corresponds to about 60% of the total phosphatase activity. 3. Two of the fractions have glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and phosphoglycerate mutase activities in a ratio similar to that of the glycerate-2,3-P2 synthase described in mammalian skeletal muscle. Their synthase activity corresponds to about 90% of the total synthase activity, and their phosphatase activity represents about 1% of the total phosphatase activity of the tissue. 4. The fourth fraction shows only glycerate-2,3-P2 phosphatase activity and represents about 40% of the total activity of the tissue. 5. It is suggested that in chicken skeletal muscle the metabolism of the glycerate-2,3-P2 is regulated in a way similar to that described in mammalian skeletal muscle.     

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.     

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.     

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.     

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.     

A specific enzyme for glucose 1,6-bisphosphate synthesis.

The reaction: glycerate-1,3-P2 PLUS GLUCOSE-1-P YIELDS TO GLUCOSE-1,6-P2 plus glycerate-P is catalyzed by a distinct enzyme of mouse brain. A divalent metal requirement was shown when the enzyme was treated with imidazole and EDTA. Mg2+, Mn2+, Ca2+, Zn2+, Ni2+, Co2+, and Cd2+ were quite effective cofactors. The enzyme, in better than 50 percent yield, has been purified away from 99 percent of the phosphoglucomutase, phosphoglycrate mutase, and phosphofructokinase. Acetyl-P, ATP, enolpyruvate-P, creatine-P, and fructose-1,6-P2 are not phosphoryl donors. Glucose-6-P and mannose-1-P are good alternate acceptors. Mannose-6-P, galactose-Ps, and fructose-Ps have little or no acceptor activity. Strong inhibition was found with fructose-1,6-P2, glycerate-2,3-P2, enolpyruvate-P, and acetyl CoA. From the amount of activity and the kinetic constants of the purified enzyme it seems likely that this enzyme is responsible for the glucose-1,6-P2 synthesis of brain.     

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).     

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.     

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.     

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.     

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.     

Multiplicity of interactions between dromedary hemoglobin and solvent components. A structural and functional study

The interaction of dromedary hemoglobin with various solvent components [2-(p-chlorophenoxy)-2-methylpropionic acid (CFA), 2,3-bisphospho-D-glycerate (glycerate-2,3-P2) and chloride] has been studied. 1. CFA greatly lowers the oxygen affinity of dromedary hemoglobin. 2. The oxygen-linked CFA binding sites are probably located in the deoxy derivative at the alpha cleft, while in the oxy form and in the presence of two other effectors (glycerate-2,3-P2 and chloride) additional, structurally and possibly functionally relevant binding site(s) should be considered. 3. Both CFA and glycerate-2,3-P2 stabilize the deoxy-like tertiary structure in the oxy derivative. 4. Chloride appears to be fundamental to obtain quaternary structural changes. 5. Interaction energy, retained in the protein when the three ligands (CFA, glycerate-2,3-P2 and chloride) are bound to the oxy form, favours intermediates not stable if only one or two allosteric effector(s) is (are) present on the protein. 6. The oxygen affinity appears to be related to both tertiary and quaternary structural changes, while cooperatively is largely invariant with solvent conditions. In conclusion, the functional properties of dromedary hemoglobin do not depend in any simple way on the variety of stabilized conformations.     

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.     

The isolation and partial characterization of diphosphoglycerate mutase from human erythrocytes.

Diphosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes. The native enzyme has a molecular weight of 57 000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of about 26 500, indicating that diphosphoglycerate mutase is comprised of two subunits of similar mass. The enzyme exhibits the following intrinsic activities: diphosphoglyceratemutase, monophosphoglycerate mutase, and 2,3-diphosphoglycerate phosphatase. The latter activity is enhanced in the presence of either organic or inorganic anions. Glycolate-2-P, particularly, has a profound activating effect. Nonspecific phosphatase and enolase activities are absent. The enzyme has an extinction coefficient at 280 nm of 1.65 cm2/mg. The amino acid composition of the homogeneous protein has been determined.     

牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究

牛蛙心脏中乳酸脱氢酶在聚丙烯酰胺凝胶电泳上显示3种同工酶区带,分别命名为LDH1、LDH2、LDH3,其中LDH1的活力占绝对优势.采用HiTrap^TM Blue HP 亲和层析和DEAE-Sephadex A离子交换层析对牛蛙骨骼肌中的LDH3进行了分离纯化.纯化的LDH3比活力为295 U/mg,Km NADH=0.028,Km丙酮酸=1.242,在SDS-PAGE上显示两条带,提示该同工酶是由两种亚基组成的,亚基的分子量分别为35.3 kD和37.6 kD.     

Immunological Difference between Glucose-6-P Dehydrogenase and Hexose-6-P Dehydrogenase from Human Liver

SHAW and Barto¹ have demonstrated the presence of an autosomally inherited glucose-6-P dehydrogenase (G6PD) in the deer mouse. Subsequently, Ohno et al.² found a similar enzyme in trout and showed that this enzyme and the autosomally inherited mouse enzyme differed from the sex-linked G6PD in possessing marked catalytic activity with galactose-6-P. This autosomally inherited G6PD was therefore named hexose-6-P dehydrogenase (H6PD)^2,3. It was shown to oxidize glucose-6-P, galactose-6-P, mannose-6-P and 2-deoxy glucose-6-P with a K_m of the order of 10^?5 M. It also oxidizes glucose with a K_m of 0.7 M³. It appears to be identical to the so-called “glucose dehydrogenase”. The enzyme utilizes both NAD and NADP and is microsome-bound. G6PD is localized in the soluble fraction of the cells of various tissues. Although it has been shown that two dehydrogenases from liver have different substrate specificity, molecular weight and elec-trophoretic mobility^3,4, it has been suggested that the two enzymes are merely isozymes and they might be interconvertible^5–7. We have now partially purified the two enzymes from human liver and show that they have different immunological properties.     

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.     

Purification of the glutamine synthetase II isozyme of Drosophila melanogaster and structural and functional comparison of glutamine synthetases I and II

Glutamine synthetase II was purified from Drosophila melanogaster adults. It was completely separable from the isozyme glutamine synthetase I by means of DEAE chromatography. The complete enzyme has an apparent molecular weight of 360,000. After two-dimensional electrophoresis it gave a single molecular species with an apparent molecular weight of 42,000. Structural analysis of the two isozymes showed that they are different both in subunit molecular weight and in isoelectric point. Peptide maps of the purified subunits showed considerable dissimilarity. Glutamine synthetase II is more active than glutamine synthetase I in the transferase assay, while the opposite is true in the biosynthetic assay. The kinetic parameters were determined, showing again noteworthy differences between the two isozymes. We therefore conclude that two forms of glutamine synthetase are present in Drosophila, with different primary structures, different kinetic behavior, and the possibility of different functional properties.     

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.     

The enzymic synthesis of ribose-1,5-bisphosphate: studies of its role in metabolism

Ribose-1,5-bisphosphate is synthesized in a reaction that uses ribose-1(or 5)-P as the phosphoryl acceptor and the acyl-P of 3-phosphoglyceryl phosphate as the donor. Glucose-1,6-bisphosphate is synthesized in a similar reaction. The relative activity with the two substrates remains unchanged over almost 300-fold purification of the enzyme, indicating that glucose-1,6-bisphosphate synthase catalyzes both reactions. The relative V/Km values for alternative phosphoryl acceptors are ribose-1-P (1); glucose-1-P (0.30); mannose-1-P and ribose-5-P (0.11); glucose-6-P (0.10); 2-deoxyglucose-6-P (0.03); and 2-deoxyribose-5-P (0.02). Fructose-1- and 6-phosphates are not substrates. The synthesis of both ribose-1,5-bisphosphate and glucose-1,6-bisphosphate is inhibited by physiologically significant levels of fructose-1,6-bisphosphate, glycerate-2,3-bisphosphate, glycerate-3-phosphate, citrate, and inorganic phosphate. Ribose-1,5-bisphosphate is a strong activator of brain phosphofructokinase.