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At least 19 recordsLinked to original sources
PubMed1987

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.

Adenosine Triphosphate↗
PubMed1978

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.

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↗
PubMed1999

Cloning and sequencing of the coenzyme B(12)-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis, reconstitution of mutase activity, and characterization of the recombinant enzyme produced in Escherichia coli.

Isobutyryl-CoA mutase (ICM) catalyzes the reversible, coenzyme B(12)-dependent rearrangement of isobutyryl-CoA to n-butyryl-CoA, which is similar to, but distinct from, that catalyzed by methylmalonyl-CoA mutase. ICM has been detected so far in a variety of aerobic and anaerobic bacteria, where it appears to play a key role in valine and fatty acid catabolism. ICM from Streptomyces cinnamonensis is composed of a large subunit (IcmA) of 62.5 kDa and a small subunit (IcmB) of 14.3 kDa. icmB encodes a protein of 136 residues with high sequence similarity to the cobalamin-binding domains of methylmalonyl-CoA mutase, glutamate mutase, methyleneglutarate mutase, and cobalamin-dependent methionine synthase, including a conserved DXHXXG cobalamin-binding motif. Using IcmA and IcmB produced separately in Escherichia coli, we show that IcmB is necessary and sufficient with IcmA and coenzyme B(12) to afford the active ICM holoenzyme. The large subunit (IcmA) forms a tightly associated homodimer, whereas IcmB alone exists as a monomer. In the absence of coenzyme B(12), the association between IcmA and IcmB is weak. The ICM holoenzyme appears to comprise an alpha(2)beta(2)-heterotetramer with up to two molecules of bound coenzyme B(12). The equilibrium constant for the ICM reaction at 30 degrees C is 1.7 in favor of isobutyryl-CoA, and the pH optimum is near 7.4. The K(m) values for isobutyryl-CoA, n-butyryl-CoA, and coenzyme B(12) determined with an equimolar ratio of IcmA and IcmB are 57 +/- 13, 54 +/- 12, and 12 +/- 2 microM, respectively. A V(max) of 38 +/- 3 units/mg IcmA and a k(cat) of 39 +/- 3 s(-1) were determined under saturating molar ratios of IcmB to IcmA.

Acyl Coenzyme A↗
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↗
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

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites.

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.

Binding Sites↗
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↗
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

Inhibition of chorismate mutase activity of chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Inhibition data (I50 values) have been obtained for inhibitors of the chorismate mutase activity of chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes. Several 1-substituted adamantane derivatives were investigated; the order of decreasing inhibitory activity with the various substituents was -PO32- much greater than -P(OCH3)O2- greater than -CO2- greater than -CH2CO2- greater than -SO2- greater than SO3-. 3-Chloroadamantane-1-acetic acid was slightly less effective than adamantane-1-acetic acid. 2-(1-Carboxy-1,4-dihydrobenzyl)acrylic acid (19), an analogue of prephenate, was an effective inhibitor. Other substances investigated, including 2,4,10-trioxaadamantane-1-acetic acid (15), 5-enolpyruvylshikimic acid (20), and 1-(carboxyethyl)-1,4-dihydrobenzoic acid (18), failed to inhibit chorismate mutase activity under the conditions investigated.

Chemical Phenomena↗
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↗
PubMed1999

Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis: influence on polyketide antibiotic biosynthesis.

The coenzyme B(12)-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis. Mutants prepared by inserting a hygromycin resistance gene (hygB) into either icmA or mutB, encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA::hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The mutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. (13)C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.

Bacterial Proteins↗
PubMed2005

Crystal structures of Mycobacteria tuberculosis and Klebsiella pneumoniae UDP-galactopyranose mutase in the oxidised state and Klebsiella pneumoniae UDP-galactopyranose mutase in the (active) reduced state.

Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the d-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH- co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25 A. The structures of Klebsiella pneumoniae mutase with FAD and with FADH- bound have been determined to 2.2 A and 2.35 A resolution, respectively. This is the first report of the FADH(-)-containing structure. Two flavin-dependent mechanisms for the enzyme have been proposed, one, which involves a covalent adduct being formed at the flavin and the other based on electron transfer. Using our structural data, we have examined the two mechanisms. The electron transfer mechanism is consistent with the structural data, not surprisingly, since it makes fewer demands on the precise positioning of atoms. A model based on a covalent adduct FAD requires repositioning of the enzyme active site and would appear to require the isoalloxazine ring of FADH- to buckle in a particular way. However, the FADH- structure reveals that the isoalloxazine ring buckles in the opposite sense, this apparently requires the covalent adduct to trigger profound conformational changes in the protein or to buckle the FADH- opposite to that seen in the apo structure.

Amino Acid Sequence↗
PubMed2004

Solvent polarity effects and limited acid catalysis in rearrangements of model radicals for the methylmalonyl-CoA mutase- and isobutyryl-CoA mutase-catalyzed isomerization reactions.

The kinetics of reactions of models for the intermediate radicals formed in the methylmalonyl-CoA mutase- and isobutyryl-CoA mutase-catalyzed rearrangements were studied by laser flash photolysis methods. The aldehyde-containing model analogous to the propanal-3-yl radical reacted via 3-exo cyclization with rate constants that varied with solvent polarity (k in the range 2 x 105 to 1 x 107 s-1). The analogous methyl ketone-containing radical reacted 2 orders of magnitude less rapidly, and the ethylthiocarbonyl-containing radical analogue reacted too slowly for kinetic measurements. No acid catalysis was observed in acetic acid, but the CF3CO2H-complexed radicals reacted 1 order of magnitude faster than the uncomplexed radicals. The results indicate that catalysis of the 3-exo radical cyclizations of the radicals formed in the enzymes by hydrogen bonding to an acid, so-called "partial protonation", is not adequate for acceleration of the reactions to the point of kinetic competence. A dissociative mechanism for the radical rearrangements in nature is considered as an alternative.

Acids↗
PubMed1994

The monofunctional chorismate mutase from Bacillus subtilis. Structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction.

Structures have been determined for chorismate mutase from Bacillus subtilis and of complexes of this enzyme with product and an endo-oxabicyclic transition state analog using multiple isomorphous replacement plus partial structure phase combination and non-crystallographic averaging. In addition to 522 water molecules, the model includes 1380 of the 1524 amino acid residues of the four trimers (each containing 3 x 127 amino acid residues) in the asymmetric unit. Refinement to 1.9 A resolution yields 0.194 for R and r.m.s. deviations from ideal values of 0.014 A for bond lengths and 2.92 degrees for bond angles. The trimer resembles a beta-barrel structure in which a core beta-sheet is surrounded by helices. The structures of the two complexes locate the active sites which are at the interfaces of adjacent pairs of monomers in the trimer. These structures have been refined at 2.2 A to a crystallographic R value of 0.18 and show r.m.s. deviations from ideal values of 0.013 A for bond lengths and 2.84 degrees or 3.05 degrees for bond angles, respectively. The final models have 1398 amino acid residues, nine prephenate molecules and 503 water molecules in the product complex, and 1403 amino acid residues, 12 inhibitor molecules and 530 water molecules in the transition state complex. The active sites of all three of these structures are very similar and provide a structural basis for the biochemical studies that indicate a pericyclic mechanism for conversion of chorismate to prephenate. The absence of reactive catalytic residues on the enzyme, the selective binding of the single reactive conformation of chorismate, the stabilization of the polar transition state, and the possible role of the C-terminal region in "capping" the active site are factors which relate these structures to the million-fold rate enhancement of this reaction.

Amino Acid Sequence↗
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↗