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PubMed1997

Magnetic field effects on coenzyme B12-dependent enzymes: validation of ethanolamine ammonia lyase results and extension to human methylmalonyl CoA mutase.

Enzymes with radical-pair intermediates have been considered as a likely target for purported magnetic field effects in humans. The bacterial enzyme ethanolamine ammonia lyase and the human enzyme methylmalonyl-CoA mutase catalyze coenzyme B12-dependent rearrangement reactions. A common step in the mechanism of these two enzymes is postulated to be homolysis of the cobalt-carbon bond of the cofactor to generate a spin-correlated radical pair consisting of the 5'-deoxyadenosyl radical and cob(II)alamin [Ado. Cbl(II)]. Thus, the reactions catalyzed by these enzymes are expected to be sensitive to an applied magnetic field according to the same principles that control radical pair chemical reactions. The magnetic field effect on ethanolamine ammonia lyase reported previously has been corroborated independently in one of the authors' laboratory. However, neither the human nor the bacterial mutase from Propionibacterium shermanii exhibits a magnetic field effect that could be greater than about 15%, considering the error limit imposed by the uncertainty of the coupled assay. Our studies suggest that putative magnetic field effects on physiological processes are not likely to be mediated by methylmalonyl-CoA mutase.

Cobamides↗
PubMed1993

Varying neurological phenotypes among muto and mut- patients with methylmalonylCoA mutase deficiency.

MethylmalonylCoA mutase (MCM) is a mitochondrial homodimer responsible for the isomerization of methylmalonylCoA to succinylCoA. Apomutase defects are traditionally divided into muto and mut- classes on the basis of residual mutase activity. Clinical findings were reviewed in 20 patients with methylmalonic aciduria secondary to MCM deficiency. All 11 muto patients had an early neonatal presentation; 6 of these patients died in infancy and 3 of 5 survivors had a poor neurological outcome as evidenced by severe delay or spastic quadriparesis with dystonia. The 2 other survivors include a 27-month-old child with a mild delay in verbal and fine motor skills and an adolescent with low normal intelligence. Of the 9 mut- patients, 7 became symptomatic in late infancy or childhood and 2 were picked up on screening. Two of the 9 patients have never had an episode of metabolic decompensation yet both are neurologically compromised; one severely retarded and autistic, the other mildly delayed. Four mut- patients have had episodic acidosis and are neurologically moderately affected, while 3 have had episodic acidosis and are neurologically intact. These results confirm phenotypic pleomorphism without a consistent pattern of neurological injury and suggest some broad correlation between mutase class and phenotype. Survival with good outcome is possible among muto patients as is significant morbidity among mut- patients. Acidosis and metabolic imbalance are not necessary preconditions for significant morbidity.

Amino Acid Metabolism, Inborn Errors↗
PubMed1999

Polyanionic inhibitors of phosphoglycerate mutase: combined structural and biochemical analysis.

The effects that the inhibitors inositol hexakisphosphate and benzene tri-, tetra- and hexacarboxylates have on the phosphoglycerate mutases from Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined. Their Kivalues have been calculated, and the ability of the inhibitors to protect the enzymes against limited proteolysis investigated. These biochemical data have been placed in a structural context by the solution of the crystal structures of S. cerevisiae phosphoglycerate mutase soaked with inositol hexakisphosphate or benzene hexacarboxylate. These large polyanionic compounds bind to the enzyme so as to block the entrance to the active-site cleft. They form multiple interactions with the enzyme, consistent with their low Kivalues, and afford good protection against limited proteolysis of the C-terminal region by thermolysin. The inositol compound is more efficacious because of its greater number of negative charges. The S. pombe phosphoglycerate mutase that is inherently lacking a comparable C-terminal region has higher Kivalues for the compounds tested. Moreover, the S. pombe enzyme is less sensititive to proteolysis, and the presence or absence of the inhibitor molecules has little effect on susceptibility to proteolysis.

Amino Acid Sequence↗
PubMed2002

Mechanistic implications for Escherichia coli cofactor-dependent phosphoglycerate mutase based on the high-resolution crystal structure of a vanadate complex.

The structure of Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), complexed with the potent inhibitor vanadate, has been determined to a resolution of 1.30 A (R-factor 0.159; R-free 0.213). The inhibitor is present in the active site, principally as divanadate, but with evidence of additional vanadate moieties at either end, and representing a different binding mode to that observed in the structural homologue prostatic acid phosphatase. The analysis reveals the enzyme-ligand interactions involved in inhibition of the mutase activity by vanadate and identifies a water molecule, observed in the native E.coli dPGM structure which, once activated by vanadate, may dephosphorylate the active protein. Rather than reflecting the active conformation previously observed for E.coli dPGM, the inhibited protein's conformation resembles that of the inactive dephosphorylated Saccharomyces cerevisiae dPGM. The provision of a high-resolution structure of both active and inactive forms of dPGM from a single organism, in conjunction with computational modelling of substrate molecules in the active site provides insight into the binding of substrates and the specific interactions necessary for three different activities, mutase, synthase and phosphatase, within a single active site. The sequence similarity of E.coli and human dPGMs allows us to correlate structure with clinical pathology.

2,3-Diphosphoglycerate↗
PubMed1991

Regulation of chorismate mutase activity of various yeast species by aromatic amino acids.

The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species. All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol. The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan. The tryptophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 microM (Yarrowia lipolytica). Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine. The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone. The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan. Six enzyme sources were not inhibited and the Y. lipolytica chorismate mutase was slightly activated by both amino acids.

Amino Acids↗
PubMed1992

Correction of methylmalonyl-CoA mutase deficiency in Mut0 fibroblasts and constitution of gene expression in primary human hepatocytes by retroviral-mediated gene transfer.

Methylmalonic acidemia is an often fatal inborn error of organic acid metabolism due to deficiency of methylmalonyl-CoA mutase. The cloning of genes encoding this enzyme and the advent of technologies for gene transfer have introduced the possibility of somatic gene therapy for this disorder. Gene therapy may require replacement of the defective enzyme in hepatocytes, which have a greater capacity for propionate metabolism than other somatic cells and represent the principle physiological site of propionate metabolism. We describe construction of an amphotropic retroviral vector containing the human methylmalonyl-CoA mutase cDNA. This vector is shown to transduce primary MCM-deficient fibroblasts and restore levels of [14C]propionate metabolism by cultures of nonselected cells to normal. This vector will transduce primary human hepatocytes and direct transcription of recombinant human MCM from the integrated provirus. This work demonstrates the feasibility of retroviral-mediated gene transfer of methylmalonyl-CoA mutase into primary human cells, including hepatocytes which represent a difficult, but potentially necessary, target for gene therapy of methylmalonic acidemia.

3T3 Cells↗
PubMed1982

Methylmalonic acidaemia due to mutase apoenzyme defect: responsive to vitamin B12 in intact fibroblasts but not in vivo.

Congenital methylmalonic acidaemia (MMA-aemia) was diagnosed in an 8-month-old girl who presented with severe metabolic acidosis, hypoglycaemia and hyperglycinaemia. Vomiting, failure to thrive and apathy first appeared when breast feeding was replaced by a cows' milk formula at the age of 3 months. The patient, unresponsive to OH-Cbl therapy, was successfully treated with dietary protein restriction and with Shohl's solution. Aged 4 years 9 months, she is in good health. Studies in cultured fibroblasts revealed a defect of the MMA-CoA mutase apoenzyme. Mutase activity in cell extracts was barely detectable both with and without added coenzyme (Ado-Cbl). Addition of OH-Cbl to the culture medium improved overall propionate metabolism in intact fibroblasts but had no effect on mutase activity in cell extracts. These observations point to the presence of a very labile mutant enzyme, suggesting that the patient reported here may be suffering from yet another variant of MMA-aemia.

Amino Acid Metabolism, Inborn Errors↗
PubMed2004

Chorismate mutase of Thermus thermophilus is a monofunctional AroH class enzyme inhibited by tyrosine.

aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/ Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-alpha/beta barrel enzyme that is organized as a trimer. Comparison of the enzyme's structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein's surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a Km of 290 microM, and a kcat/ Km value of 180 s(-1) mM(-1). TtCM was inhibited by tyrosine with a Ki =34 microM, possibly in a competitive manner.

Amino Acid Sequence↗
PubMed1986

Chorismate mutase isoenzymes from Sorghum bicolor: immunological characterization.

Highly purified fractions of chorismate mutase 1 and 2 from etiolated seedlings of Sorghum bicolor were used as the antigen for antibody production in BALB/c mice. Tests for antigen-antibody complex formation were made by immunodiffusion, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). These tests indicated the presence of specific antibodies for each isoenzyme in their antisera. However, in the same tests, no cross-reaction was found between chorismate mutase 1 and 2 and their antisera. This indicates no immunological similarity between the two isoenzymes of chorismate mutase from sorghum.

Animals↗
PubMed1992

Monofunctional chorismate mutase from Serratia rubidaea: a paradigm system for the three-isozyme gene family of enteric bacteria.

Serratia rubidaea (ATCC 27614) typifies a substantial number of enteric bacteria which, unlike Escherichia coli, possess a monofunctional species of chorismate mutase (denoted CM-F). CM-F coexists with two additional species of chorismate mutase, each of the latter being one catalytic domain of a bifunctional protein. The two bifunctional proteins are utilized for phenylalanine (CM-P/prephenate dehydratase) and tyrosine (CM-T/cyclohexadienyl dehydrogenase) biosynthesis in all enteric bacteria. S. rubidaea was selected as the organism of choice for purification of CM-F because of the relatively abundant level of expression found for this enzyme. The monofunctional CM-F enzyme was purified about 1600-fold with a yield of about 16%. This is the first monofunctional chorismate mutase to be purified from any gram-negative prokaryote. The CM-F enzyme is a positively charged homodimer made up of 20-kDa subunits. It has a pH optimum of 5.5, exhibits a Km value of 0.33 mM for chorismate, and is sensitive to product inhibition by prephenate that is competitive with respect to chorismate. It is insensitive to feedback inhibition by any of the aromatic amino acids. Partial purification of the bifunctional P-protein and the bifunctional T-protein was also carried out in order to compare the properties of CM-F, CM-P, and CM-T in a common organism. The most striking differential properties of the three isozymes were those of pH optimum and degree of protection conferred by dithiothreitol.

Binding, Competitive↗
PubMed1985

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).

Animals↗
PubMed1989

Sequence of rat skeletal muscle phosphoglycerate mutase cDNA.

A cDNA clone coding rat skeletal muscle phosphoglycerate mutase was isolated from a rat muscle lambda gt10 cDNA library and its sequence was determined. The deduced protein possesses 252 amino acids and is 94% homologous with respect to human muscle phosphoglycerate mutase. No amino acids changes occur at the active site and structural predictions suggest strong conformational homologies with other enzymes of the mutase family.

Amino Acid Sequence↗
PubMed1989

Assay of methylmalonyl CoA mutase with high-performance liquid chromatography.

An assay for methylmalonyl CoA mutase activity is described. Succinyl CoA produced in this method is separated from the substrate, methylmalonyl CoA, by reverse-phase high-performance liquid chromatography and is quantified. This method is useful to differentiate mutase apoenzyme deficiency (mut0, mut-) and the defect in deoxyadenosylcobalamin synthesis using fibroblasts cultured in high concentration of supplementary hydroxocobalamin. In methylmalonic acidemia, measurement of lymphocytes mutase activity offers therapeutical and prognostic informations.

Acyl Coenzyme A↗
PubMed1982

Effect of vanadate on the formation and stability of the phosphoenzyme forms of 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and of phosphoglucomutase.

2,3-Bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-bisphospho-D-glycerate:2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) and phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate:alpha -D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1), which are markedly inhibited by vanadate, possess a ping-pong mechanism involving an intermediate phosphoenzyme. The formation and the stability of these phosphoenzymes have been examined spectrophotometrically in the absence of vanadate. Vanadate does not inhibit the phosphorylation of either mutase by its cofactor. The instability of the phosphoenzyme form of phosphoglycerate mutase increases in the presence of vanadate, but the stability of the phosphorylated phosphoglucomutase is not affected.

2,3-Diphosphoglycerate↗
PubMed1993

A tyrosine residue is involved in the allosteric binding of tryptophan to yeast chorismate mutase.

Comparative 1H-NMR studies have been carried out on wild-type chorismate mutase, which is activated by tryptophan and inhibited by tyrosine and phenylalanine, and mutant yeast chorismate mutase, which has a single point mutation (T226I) and is not allosterically regulated but 'locked' in the activated state. Double quantum-filtered COSY spectra show cross-peaks which have been assigned to a tyrosine that are absent in the mutant enzyme and in the wild-type enzyme plus tryptophan when compared with the wild-type enzyme alone. These observations indicate the involvement of a tyrosine at or near the allosteric binding site. The involvement of tyrosine in tryptophan binding was tested by modification of tyrosine in yeast chorismate mutase by nitration with tetranitromethane. All forms of the enzyme exhibited an approx. 50% reduction in specific activity, but it was found that preincubation of the wild-type with the allosteric activator, tryptophan, lead to partial protection against loss in specific activity. Only one tyrosine residue was nitrated in the wild-type enzyme and this tyrosine was identified by tryptic digestion and sequencing, and found to be very close to the site of the single point mutation in the mutant enzyme. It is proposed that Tyr-234 is located at or near the allosteric activation site.

Amino Acid Sequence↗
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↗
PubMed1996

Distribution of phosphoglycerate mutase isozymes in rat, rabbit and human tissues.

The distribution of phosphoglycerate mutase isozymes (types MM, MB and BB) in rat, rabbit and human tissues has been studied by electrophoresis on cellulose acetate and by highly-resolutive ion exchange chromatography. In the three species, muscle is the tissue with higher phosphoglycerate mutase activity. Heart is the only tissue with the three phosphoglycerate mutase isozymes in substantial amounts. Skeletal muscle contains mostly type MM isozyme and the other tissues possess almost exclusively type BB isozyme. Even in the presence of inhibitors, adenylate kinase can interfere with the staining reactions when large samples are analyzed and a long period of incubation is required.

Adenylate Kinase↗