PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “MUTASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources
PubMed1997

A base-off analogue of coenzyme-B12 with a modified nucleotide loop--1H-NMR structure analysis and kinetic studies with (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase.

(Co beta-5'-Deoxyadenosin-5'-yl)-(p-cresolyl)cobamide (Ado-PCC), an analogue of the base-off form of coenzyme-B12 (CoB12), was prepared by alkylation of (Co alpha/beta-cyano/aqua)-(p-cresolyl)cobamide (PCC) with 5'-chloro-5'-deoxyadenosine. The 500 MHz 1H-NMR spectrum of Ado-PCC in D2O at pH 7.4 was completely analyzed using COSY and NOESY two-dimensional experiments. The coenzyme and inhibitory activities of Ado-PCC were tested with three coenzyme-B12-dependent enzymes: (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase. Ado-PCC showed strong coenzyme activity with methylmalonyl-CoA mutase, which is known to bind the base-off form of CoB12. In contrast, Ado-PCC had no coenzyme activity but acted instead as a competitive inhibitor with glycerol dehydratase and diol dehydratase, which are likely to prefer the base-on form of CoB12. These results indicate that Ado-PCC, whose structure is analogous to the base-off form of CoB12, can be used for probing the mode of coenzyme binding by coenzyme-B12-dependent enzymes.

Cobamides↗
PubMed2001

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from Trypanosoma brucei: metal-ion dependency and phosphoenzyme formation.

Recombinant cofactor-independent phosphoglycerate mutase from Trypanosoma brucei was inactivated by EDTA, and reactivated by Co(2+) much more than by Mn(2+) or Fe(2+). It displayed a minor phosphoglycerate phosphatase activity, which was stimulated by Mn(2+) more than by Co(2+). Upon incubation with [(32)P]phosphoglycerate, radioactivity was incorporated into the enzyme, most particularly in the presence of Mn(2+) or Fe(2+). The phosphorylated residue was identified by tandem mass spectrometry as Ser74, a residue homologous to the phosphorylated serine in alkaline phosphatase. However, the rates of formation and of disappearance of this phosphoenzyme were quite low compared to the mutase reaction. This and other properties indicated that the observed phosphoenzyme is an intermediate in the minor phosphatase activity rather than in the phosphomutase reaction.

2,3-Diphosphoglycerate↗
PubMed1981

Human muscle phosphoglycerate mutase deficiency: newly discovered metabolic myopathy.

Muscle phosphoglycerate mutase activity was decreased (5.7 percent of the lowest control value) in a 52-year-old man with intolerance for strenuous exercise and recurrent pigmenturia since adolescence. All of the other enzymes of glycolysis had normal activities, and glycogen concentration was normal. Electrophoretic, heat lability, and mercury inhibition studies showed that the small residual activity in the patient's muscle was represented by the brain (BB) isoenzyme of phosphoglycerate mutase, suggesting a genetic defect of the M subunit which predominates in normal muscle. The prevalence of the BB isoenzyme in other tissues, including muscle culture, may explain why symptoms were confined to muscle.

Brain↗
PubMed1995

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2006

Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids.

The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 +/- 0.05 mM and a k(cat) of 60 s(-1) per active site (at 37 degrees C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 A resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.

Amino Acids, Aromatic↗
PubMed1979

Purification and properties of the manganese-dependent phosphoglycerate mutase of Bacillus subtilis.

Phosphoglycerate mutase of Bacillus subtilis was purified to apparent homogeneity. It specifically required manganese ions for stability and activity, but it does not need 2,3-diphosphoglycerate as cofactor; the Km for Mn2+ is about 4.5 micrometer. Enzyme activity was inhibited by heavy-metal ions, 2,3-butanedione, and sulfhydryl agents. The mutase has a molecular weight of about 74,000 as shown by Sephadex gel filtration and by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; it consisted of one polypeptide.

Amino Acids↗
PubMed2002

Inactivation of phosphoglycerate mutase and creatine kinase isoenzymes in human serum.

AIMS/BACKGROUND: Total phosphoglycerate mutase (PGM) activity in serum has been shown to be increased in acute myocardial infarction with the same time course as creatine kinase (CK) activity. However, the increase in the muscle (MM) and in the cardiac (MB) PGM isoenzymes was not as high as expected. The present study was undertaken to characterise PGM inactivation by serum and to compare it with serum CK inactivation. METHODS: The PGM and the CK activities of extracts of human heart, skeletal muscle, and brain were determined spectrophotometrically after incubation with different media, namely: plasma, whole serum, dialysed serum, heated serum, serum ultrafiltrate, urate solution, and buffer solution. RESULTS: Type MM PGM was inactivated by plasma, whole serum, heated serum, dialysed serum, and serum ultrafiltrate. Inactivation in dialysed serum was reduced by EDTA and largely reversed by thiol agents. Inactivation in serum ultrafiltrate was not prevented by EDTA and only partially reversed by dithiothreitol. The muscle and type BB CK isoenzymes were inactivated in all the tested media. The incubation of human and rabbit skeletal muscle PGM and CK in urate solution showed that urate does not affect mutase activity under conditions that inactivate CK. CONCLUSIONS: These results confirm the mechanisms of CK inactivation proposed by others and show that the type M PGM subunit is inactivated by two different mechanisms, which appear to involve the thiol groups of the enzyme. One mechanism is caused by either a protein component or a protein bound serum component and involves calcium ions and/or another chelatable metal ion. The other mechanism is caused by a lower molecular weight serum component and is metal ion independent.

Animals↗
PubMed1976

Congenital methylmalonic acidemia: a variant of the B12 'non-responsive' form with evidence for reduced affinity of methylmalonyl-CoA mutase for its B12-coenzyme.

Methylmalonate metabolism was investigated in fibroblasts and leukocytes of two unrelated patient with a B12-nonresponsive type of congenital methylmalonic acidemia. Intact fibroblasts from both patients showed a defective metabolism of methyl-14 c-malonate to 14CO2, whereas no such defect was found in their intact peripheral leukocytes. In disrupted fibroblasts, the conversion of methylmalonyl coenzyme A to succinyl coenzyme A was markedly reduced but was completely normalized by the addition of 5'-deoxyadenosylcobalamin (AdoCb1; 10(-5) mol/l), the specific coenzyme of methylmalonyl coenzyme A mutase. Assays with decreasing concentrations of AdoCbl (10(-5)-10(-11) mol/l) suggested a reduced affinity of the mutase apoenzyme for its coenzyme, implicating yet another variant of this heterogeneous disease.

Amino Acid Metabolism, Inborn Errors↗
PubMed2001

The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa.

BACKGROUND: Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. RESULTS: The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. CONCLUSIONS: Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously.

Amino Acid Sequence↗
PubMed1988

Inactivation of type MM phosphoglycerate mutase by sulfhydryl group reagents during facial embryogenesis.

Type MM phosphoglycerate mutase from free dissected mandibular processes from embryonic rats was reversibly inactivated by tetrathionate, p-chloromercuribenzoate, and Hg2+. Titration with p-chloromercuribenzoate showed the existence of two sulfhydryl groups per enzyme subunit, the modification of which produced a progressive decline in enzyme activity. The apparent Km values for substrate and cofactor were not affected by tetrathionate treatment. Phosphoglycerate mutase inactivated by tetrathionate and by p-chloromercuribenzoate was unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P2. Glycerate-2,3-P2 protected against tetrathionate but failed to protect against Hg2+ and p-chloromercuribenzoate.

Animals↗
PubMed1976

Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.

Affinity Labels↗
PubMed1991

Yeast expression of a catalytic antibody with chorismate mutase activity.

The catalytic antibody 1F7 promotes the rearrangement of chorismate into prephenate. We cloned and sequenced the genes encoding this catalyst to determine the origin of the observed rates and specificity. The antibody cDNAs were modified and inserted into inducible expression vectors. Simultaneous intracellular expression of the light and truncated heavy chains in strains of the yeast Saccharomyces cerevisiae lacking natural chorismate mutase resulted in the production of properly folded and assembled Fab antibody. Assembly of the light and heavy immunoglobulin chains is roughly 60-70% efficient in our in vivo system, lagging behind light chain synthesis throughout log and stationary phase. Nevertheless, high intracellular levels of functional Fab antibody (0.1% of total cellular protein) were obtained with an ultra-high copy number plasmid. As yeast-derived 1F7(Fab) catalyzes the chorismate mutase reaction with the same specific activity as antibody isolated from the hybridoma, our expression system now makes possible the application of classical and "reverse" genetics to the study and improvement of this first-generation abzyme.

Amino Acid Sequence↗
PubMed1989

Structure of the gene encoding the muscle-specific subunit of human phosphoglycerate mutase.

We report the isolation and analysis of genomic clones containing the entire gene encoding the muscle-specific subunit of human phosphoglycerate mutase. The gene spans 2.83 kilobase pairs and has a three-exon/two-intron structure that is similar to the organization of the human 2,3-bisphosphoglycerate mutase gene (Joulin, V., Garel, M.-C., LeBoulch, P., Valentin, C., Rosa, R., Rosa, J., and Cohen-Solal, M. (1988) J. Biol. Chem. 263, 15785-15790), in that the second introns of both genes are localized precisely at the same position. A canonical TATA box and an inverted CCAAT box are present immediately upstream of this gene. Comparison with other muscle-specific enzyme genes reveals a conserved 9-base pair element (GGGGCTGGG) in the 5'-flanking region that may be associated with the expression of genes encoding muscle-specific enzymes.

Amino Acid Sequence↗
PubMed1987

Isolation of a cDNA encoding the muscle-specific subunit of human phosphoglycerate mutase.

We have isolated a full-length cDNA specifying the muscle-specific subunit of human phosphoglycerate mutase (PGAM-M). The cDNA encodes a deduced protein 253 amino acids in length and contains an unusually short (37 nucleotides) 3'-untranslated region. The deduced human PGAM-M protein is clearly related to yeast PGAM and to human diphosphoglycerate mutase. Genomic Southern analysis using the PGAM-M cDNA as a probe implies the presence of a large PGAM gene family in the human genome, while Northern analysis demonstrates tissue-specific transcription of this isoenzyme gene.

Amino Acid Sequence↗
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↗
PubMed1984

Spectrophotometric assay of bisphosphoglycerate mutase: a reexamination of Rapoport-Luebering's method.

The saturation by substrates and cofactors, the effects of pH and the influence of salts and auxiliary enzymes have been studied. The linear NAD+ reduction observed before addition of haemolysate to the assay system was proportional to pH, being higher with phosphate than with Tris-HCl buffer. In the presence of bisphosphoglycerate mutase, an optimal pH (7.8-8.1) was obtained and the inhibition by sulfate ions could be confirmed. It can then be suggested that the absence of an equilibrium, the pH used by several authors and sulfate inhibition could be sources of error in the spectrophotometric assay of bisphosphoglycerate mutase activity. Once optimal conditions have been established, activities found in both human and rat erythrocytes are similar to those given by other accurate methods.

Animals↗