Information on EC 5.4.99.2 - Methylmalonyl-CoA mutase and Organism(s) Homo sapiens

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Homo sapiens


The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea


The taxonomic range for the selected organisms is: Homo sapiens

EC NUMBER
COMMENTARY hide
5.4.99.2
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RECOMMENDED NAME
GeneOntology No.
Methylmalonyl-CoA mutase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-methylmalonyl-CoA = succinyl-CoA
show the reaction diagram
mechanism, overview, a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for Co-C bond homolysis, key role for the conserved polar glutamate residue in controlling the initial generation of radical species
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
isomerization
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
3-hydroxypropanoate cycle
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3-hydroxypropanoate/4-hydroxybutanate cycle
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anaerobic energy metabolism (invertebrates, mitochondrial)
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conversion of succinate to propanoate
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crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle (engineered)
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methylaspartate cycle
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propanoyl CoA degradation I
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pyruvate fermentation to propanoate I
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CO2 fixation in Crenarchaeota
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propionate fermentation
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Valine, leucine and isoleucine degradation
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Glyoxylate and dicarboxylate metabolism
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Propanoate metabolism
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Carbon fixation pathways in prokaryotes
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
(R)-methylmalonyl-CoA CoA-carbonylmutase
Requires a cobamide coenzyme.
CAS REGISTRY NUMBER
COMMENTARY hide
9023-90-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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metabolic disorder methylmalonic aciduria can be caused by nonsense mutations within the methylmalonyl-CoA mutase gene, resulting in the production of a truncated protein with little or no catalytic activity
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-2-Methyl-3-oxopropanoyl-CoA
?
show the reaction diagram
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-
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(R)-2-methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
(R)-methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
(S)-methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
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-
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?
ethylmalonyl-CoA
methylsuccinyl-CoA
show the reaction diagram
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reacts about 1000-10000 times moreslowly than the natural substrate
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additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-2-Methyl-3-oxopropanoyl-CoA
?
show the reaction diagram
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-
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(R)-methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
adenosylcobalamin
Cobalamin
cobamide
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cl-
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stimulates
PO43-
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required, maximal activity at 50 mM
SO42-
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stimulates
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R)-2-methyl-3-oxopropanoyl-CoA
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substrate inhibition
Ca2+
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at high concentrations
Cyclopropylcarbonyl-CoA carboxylate
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reversible mixed-type inhibition
Methylenecyclopropylacetyl-CoA
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reversible mixed-type inhibition
methylmalonyl-CoA
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reversible mixed-type inhibition
Mg2+
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at high concentrations
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
adenosylcobalamin
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required
methylmalonic acidemia protein
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after 60 min of reaction, when MCM is inactive, the addition of methylmalonic acidemia protein increases the enzymatic activity through GTP hydrolysis, indicating reactivation of MCM by exchange of the damaged cofactor. Methylmalonic acidemia protein acts as a chaperone of human MCM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
(R)-2-methyl-3-oxopropanoyl-CoA
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0.06 - 0.2
(R/S)-2-methyl-3-oxopropanoyl-CoA
additional information
additional information
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pre-steady-state and steady-state kinetics of wild-type and mutant enzymes
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 3.95
(R)-methylmalonyl-CoA
150 - 283
(R/S)-2-methyl-3-oxopropanoyl-CoA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.33
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67.4
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
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7 - 9
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8.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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peripheral blood lymphocyte
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Homo sapiens;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
77500
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2 * 77500, SDS-PAGE
80000
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x * 80000, SDS-PAGE
144000
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gel filtration
145000
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gel filtration
150000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 80000, SDS-PAGE
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme in the apo, holo, and substrate-bound ternary forms, sitting drop vapor diffusion method, using 1.6 M Na/K-phosphate, 0.1 M HEPES pH 7.5 (apo form), or 30% (w/v) PEG3350, 0.1 M Bis-Tris pH 5.5, 0.3 M (NH4)2SO4 (holo form), or 20% (w/v) PEG3350, 0.1 M Bis-Tris pH 5.5, 0.1 M (NH4)2SO4 (ternary form)
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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15 min, stable below
55
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t1/2: 3 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
repeated freezing and thawing results in a gradual loss of activity
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 1.5 mg protein /ml in 50 mM potassium phosphate, pH 7.4, stable for at least 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Saccharomyces cerevisiae
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IMAC column chromatography
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Ni-NTA column chromatography, Resource Q column chromatography, and Superdex 200 gel filtration
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta(DE3)pLysS cells by nickel affinity and anion exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3)R3-Rosetta cells
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expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta(DE3)pLysS cells
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expression of the enzyme gene carrying a stop-codon mutation in mouse primary fibroblast cell lines, effects of gentamicin and PTC124 for stop-codon read-through potential, overview. Without treatment the cells contain 19% of the normal levels of methylmalonyl-CoA mutase enzyme activity which increases to 32% with treatment, suggesting a functional improvement. Treatment with PTC124 increases the amount of human methylmalonyl-CoA mutase gene mRNA by 1.6fold
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functional transgenic enzyme expression in enzyme-deficient Mus musculus using a AAV8-CBA-MUT vector
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mutant enzymes G94V, Y231N, R369H, G623R, H678R and G717V from patients suffering from the mut-form of methylmalonic acidemia, expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E392A
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site-directed mutagenesis, kcat is reduced 12fold compared to the wild-type enzyme. The mutant shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
E392D
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site-directed mutagenesis, kcat is reduced 330fold compared to the wild-type enzyme. The mutant shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
E392Q
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site-directed mutagenesis, kcat is reduced 16fold compared to the wild-type enzyme. The mutant shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
G623R
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six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V are detected in L-methylmalonyl-CoA mutase cDNA of patients suffering from the mut-form of methylmalonic acidemia resulting from defective adenosylcobalamin binding. The mutations increase the Km for adenosylcobalamin by 40fold to 900fold, while the values for maximal velocity varies from 0.2% to nearly 100% of that of the wild-type protein
G717V
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six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V are detected in L-methylmalonyl-CoA mutase cDNA of patients suffering from the mut-form of methylmalonic acidemia resulting from defective adenosylcobalamin binding. The mutations increase the Km for adenosylcobalamin by 40fold to 900fold, while the values for maximal velocity varies from 0.2% to nearly 100% of that of the wild-type protein
G94V
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six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V are detected in L-methylmalonyl-CoA mutase cDNA of patients suffering from the mut-form of methylmalonic acidemia resulting from defective adenosylcobalamin binding. The mutations increase the Km for adenosylcobalamin by 40fold to 900fold, while the values for maximal velocity varies from 0.2% to nearly 100% of that of the wild-type protein
H678R
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six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V are detected in L-methylmalonyl-CoA mutase cDNA of patients suffering from the mut-form of methylmalonic acidemia resulting from defective adenosylcobalamin binding. The mutations increase the Km for adenosylcobalamin by 40fold to 900fold, while the values for maximal velocity varies from 0.2% to nearly 100% of that of the wild-type protein
R369H
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six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V are detected in L-methylmalonyl-CoA mutase cDNA of patients suffering from the mut-form of methylmalonic acidemia resulting from defective adenosylcobalamin binding. The mutations increase the Km for adenosylcobalamin by 40fold to 900fold, while the values for maximal velocity varies from 0.2% to nearly 100% of that of the wild-type protein
Y231N
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six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V are detected in L-methylmalonyl-CoA mutase cDNA of patients suffering from the mut-form of methylmalonic acidemia resulting from defective adenosylcobalamin binding. The mutations increase the Km for adenosylcobalamin by 40fold to 900fold, while the values for maximal velocity varies from 0.2% to nearly 100% of that of the wild-type protein
additional information
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neither a species barrier to mitochondrial processing nor an apparent immune response to MUT limits the murine model as an experimental platform to test the efficacy of human gene therapy vectors for methylmalonic acidemia
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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method for separation of methylmalonyl-CoA and succinyl-CoA by capillary electrophoresis suitable for evaluation of total and holo-enzyme activity in biological matrices. Application of method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects
medicine
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method for separation of methylmalonyl-CoA and succinyl-CoA by capillary electrophoresis suitable for evaluation of total and holo-enzyme activity in biological matrices. Application of method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects