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At least 19 recordsLinked to original sources
PubMed1989

Long-lived tryptophan fluorescence in phosphoglycerate mutase.

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.

Animals↗
PubMed1999

Structure of a phosphoglycerate mutase:3-phosphoglyceric acid complex at 1.7 A.

The crystal structure of the tetrameric glycolytic enzyme phosphoglycerate mutase from the yeast Saccharomyces cerevisiae has been determined to 1.7 A resolution in complex with the sugar substrate. The difference map indicates that 3-phosphoglycerate is bound at the base of a 12 A cleft, positioning C2 of the substrate within 3.5 A of the primary catalytic residue, histidine 8.

Binding Sites↗
PubMed1982

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.

Animals↗
PubMed1982

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.

Animals↗
PubMed1983

Metabolism of glycerate-2,3-P2--V. Histidine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase activities of rabbit muscle phosphoglycerate mutase.

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-P2 synthase and glycerate-2,3-P2 phosphatase. The synthase and the phosphatase activities are less affected than the mutase activity. Glycerate-2,3-P2 and glycerate-3-P protect against diethylpyrocarbonate inactivation, but do not protect against inactivation produced by photo-oxidation. Hydroxylamine reactivates the diethylpyrocarbonate-treated enzyme. Chemical modification of phosphoglycerate mutase markedly reduces its ability to form the functionally active phosphoenzyme. 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.

2,3-Diphosphoglycerate↗
PubMed1984

Metabolism of glycerate-2,3-P2--VI. Lysyl-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase activities of rabbit muscle phosphoglycerate mutase.

Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with pyridoxal-5'-phosphate produces the concurrent loss of the three activities of the enzyme: phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P2. Substrate (glycerate-3-P) protects against pyridoxal-5'-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P2) does not prevent inactivation. 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. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.

Animals↗
PubMed1983

Metabolism of glycerate-2,3-P2-III. Arginine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase activities of rabbit muscle phosphoglycerate mutase.

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-P2 synthase and glycerate-2,3-P2 phosphatase. Hydroxylamine reactivates all the activities of the modified enzyme. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P2. Both substrate and cofactor protect against inactivation. 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. In addition, they suggest that arginine is involved in the binding of the cofactor to the enzyme.

Animals↗
PubMed1987

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.

Animals↗
PubMed1975

First determination of the isozyme patterns of phosphoglycerate mutases (E.C.2.7.5.3) and phosphoglycerate kinases (E.C.2.7.2.3) in human tissues.

We present in this paper the first report about identification of several fractions of phosphoglycerate mutase (PGlyM) activity using starch gel electrophoresis and two different buffer systems. A typical muscle form of PGlyM was detected. It is also shown that isozymes of phosphoglycerate kinase (PGK) can be separated through the buffer system used by Spencer et al; (1964) for the phosphogluco mutase.

Buffers↗
PubMed1989

The phosphoglycerate mutases.

The phosphoglycerate mutase family is generally very well documented with respect to structure, evolution, and mode of action. However, a few individuals in the family remain relatively poorly characterized and will clearly require more detailed study. Furthermore, certain aspects of the detailed behavior of these enzymes are, as yet, incompletely understood and require further investigation. Cofactor-dependent monophosphoglycerate mutase and bisphosphoglycerate mutase are undoubtedly very closely related. Their amino acid sequences are strongly similar, they can form active heterodimers, and they catalyze the same three reactions, albeit at substantially different relative rates. Both enzymes catalyze a ping-pong type of reaction with a phosphohistidine intermediate. The presence of an additional phospho ligand at the active site of monophosphoglycerate mutase helps to explain why this enzyme is better at retaining the 2,3-bisphosphoglycerate intermediate and why it is thus more efficient (by a factor of about 10(3)) at catalyzing the interconversion of 3- and 2-phosphoglycerates. The reason why 1,3-bisphosphoglycerate is a better substrate for bisphosphoglycerate mutase than for monophosphoglycerate mutase (by a factor of about 30) is not yet apparent but presumably relates to the relative positioning of the two phospho-binding sites. Both enzymes are equally good as phosphatases when the reaction is activated by 2-phosphoglycollate. Available evidence indicates that these mutases are similar in many respects to the much smaller, cofactor-dependent monophosphoglycerate mutase from Schizosaccharomyces pombe, but further information is required to define the relationship more precisely. Cofactor-independent monophosphoglycerate mutase belongs to a quite distinct branch of the phosphoglycerate mutase family. It is not known at present whether this branch is related divergently or convergently to the cofactor-dependent monophosphoglycerate mutase/bisphosphoglycerate mutase branch. Existing evidence can be argued both ways. For example, the kinetic evidence shows a ping-pong type of reaction and would be consistent with a phosphohistidine intermediate as encountered in the other mutases. Thus the cofactor-independent enzyme may also have arisen by gene duplication--but, in this case, yielding an enzyme of about twice the size, with slightly different residues at the active site and C-terminal tail. An alternative possibility, of course, is that the two branches of the phosphoglycerate mutase family are quite unrelated in a divergent sense and are little more similar structurally than is, for example, the catalytically similar enzyme phosphoglucomutase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Phylogeny and ontogeny of the phosphoglycerate mutases--IV. Distribution of glycerate-2,3-P2 dependent and independent phosphoglycerate mutases in algae, fungi, plants and animals.

1. The distribution of the glycerate-2,3-P2 dependent and independent phosphoglycerate mutases (PGM) has been studied in more than eighty species. 2. PGM activity in the extracts has been measured in the presence and in the absence of glycerate-2,3-P2, at pH 7.5 and at pH 8.5. 3. All samples with glycerate-2,3-P2 dependent PGM possess higher activity at pH 7.5 than at pH 8.5. In contrast, samples with glycerate-2,3-P2 independent PGM possess lower activity at pH 7.5 than at pH 8.5. 4. In algae and fungi both glycerate-2,3-P2 dependent and independent PGM have been found. 5. In plants only glycerate-2,3-P2 independent PGM has been detected. 6. In animals both types of PGM are present. Independent PGM activity is present in sponges, coelenterates, myriapods, arachnids and echinoderms. Glycerate-2,3-P2 dependent PGM is present in platyhelminths, mollusks, annelids, crustaceans, insects and vertebrates.

2,3-Diphosphoglycerate↗
PubMed1977

Phosphoglycerate mutase from wheat germ: studies with isotopically labeled 3-phospho-D-glycerates showing that the catalyzed reaction is intramolecular. Appendix: phosphoglycerate mutase from wheat germ: isolation, crystallization, and properties.

The isomerization of 3-phospho-D-glycerate and 2-phospho-D-glycerate catalyzed by the cofactor-independent phosphoglycerate mutase from wheat germ (the isolation and crystallization of which is described in the Appendix) has been shown to be intramolecular by two methods. Mass-spectrometric analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[18O]phospho-D-glycerate shows that there is no exchange of labeled phosphoryl group between carbon skeletons in the mutase-catalyzed reaction. Analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[32p]phospho-D-glycerate by a method involving the kinetic discrimination between 2(-2)H and 2(-1)H species using the enolase isotope effect similarly shows that the wheat germ phosphoglycerate mutase mediates an intramolecular transfer of the phosphoryl group.

Adenosine Triphosphate↗
PubMed1986

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.

2,3-Diphosphoglycerate↗
PubMed2000

Trypanosoma brucei contains a 2,3-bisphosphoglycerate independent phosphoglycerate mutase.

Assays of phosphoglycerate mutase (PGAM) activity in lysates of bloodstream form Trypanosoma brucei appeared not to require exogenous 2,3-bisphosphoglycerate, thus suggesting that this protist contains an enzyme belonging to the class of cofactor-independent PGAMs. A gene encoding a polypeptide with motifs characteristic for this class of enzymes was cloned. The predicted T. brucei PGAM polypeptide contains 549 amino acids, with Mr 60 557 and pI 5.5. Comparison with 15 cofactor-independent PGAM sequences available in databases showed that the amino-acid sequence of the trypanosome enzyme has 59-62% identity with plant PGAMs and 29-35% with eubacterial enzymes. A low 28% identity was observed with the only available invertebrate sequence. The trypanosome enzyme has been expressed in Escherichia coli, purified to homogeneity and subjected to preliminary kinetic analysis. Previous studies have shown that cofactor-dependent and -independent PGAMs are not homologous. It has been inferred that the cofactor-independent PGAMs are in fact homologous to a family of metalloenzymes containing alkaline phosphatases and sulphatases. Prediction of the secondary structure of T. brucei PGAM and threading the sequence into the known crystal structure of E. coli alkaline phosphatase (AP) confirmed this homology, despite the very low sequence identity. Generally, a good match between predicted (PGAM) and actual (AP) secondary structure elements was observed. In contrast to trypanosomes, glycolysis in all vertebrates involves a cofactor-dependent PGAM. The presence of distinct nonhomologous PGAMs in the parasite and its human host offers great potential for the design of selective inhibitors which could form leads for new trypanocidal drugs.

2,3-Diphosphoglycerate↗
PubMed2006

Purification, crystallization and preliminary X-ray crystallographic analysis of the archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3.

Phosphoglycerate mutases catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways. The archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to a resolution of 2.2 angstroms has been collected and processed in space group R32. Assuming the presence of a dimer in the asymmetric unit, the V(M) value is calculated to be 3.0 angstroms3 Da(-1), consistent with the dynamic light-scattering experiment result, which shows a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of Bacilllus stearothermophilus phosphoglycerate mutase as a search model did not provide a satisfactory solution, indicating substantially different structures of these two phophoglycerate mutases.

Archaeal Proteins↗
PubMed1976

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.

Animals↗