Information on EC 5.4.99.2 - Methylmalonyl-CoA mutase

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

EC NUMBER
COMMENTARY hide
5.4.99.2
-
RECOMMENDED NAME
GeneOntology No.
Methylmalonyl-CoA mutase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-methylmalonyl-CoA = succinyl-CoA
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
group transfer
-
-
intramolecular
-
isomerization
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
3-hydroxypropanoate cycle
-
-
3-hydroxypropanoate/4-hydroxybutanate cycle
-
-
anaerobic energy metabolism (invertebrates, mitochondrial)
-
-
Carbon fixation pathways in prokaryotes
-
-
CO2 fixation in Crenarchaeota
-
-
conversion of succinate to propanoate
-
-
Glyoxylate and dicarboxylate metabolism
-
-
Metabolic pathways
-
-
methylaspartate cycle
-
-
Microbial metabolism in diverse environments
-
-
Propanoate metabolism
-
-
propanoyl CoA degradation I
-
-
propionate fermentation
-
-
pyruvate fermentation to propanoate I
-
-
Valine, leucine and isoleucine degradation
-
-
SYSTEMATIC NAME
IUBMB Comments
(R)-methylmalonyl-CoA CoA-carbonylmutase
Requires a cobamide coenzyme.
CAS REGISTRY NUMBER
COMMENTARY hide
9023-90-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
Burkholderia fungorum
-
-
-
Manually annotated by BRENDA team
Baby hamster kidney fibroblast like cells
-
-
Manually annotated by BRENDA team
strain Z
Uniprot
Manually annotated by BRENDA team
strain Z
Uniprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
A4YEG1: catalytic subunit of methylmalonyl-CoA mutase, A4YIE3: coenzyme B12-binding subunit of methylmalonyl-CoA mutase
A4YEG1 and A4YIE3
UniProt
Manually annotated by BRENDA team
A4YEG1: catalytic subunit of methylmalonyl-CoA mutase, A4YIE3: coenzyme B12-binding subunit of methylmalonyl-CoA mutase
A4YEG1 and A4YIE3
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain H37Rv (ATCC 25618)
-
-
Manually annotated by BRENDA team
strain H37Rv (ATCC 25618)
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain CKD1119
-
-
Manually annotated by BRENDA team
strain CKD1119
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
additional information
no activity in Solanum tuberosum
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
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
physiological function
additional information
-
optimization and validation of a reversed-phase high performance liquid chromatography method to evalidate MCM activity in bovine liver, conditions to optimize reproducibility of the method and to determine stability of the enzyme and its product during storage and processing of samples, overview
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
(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
-
-
-
-
?
4-Carboxy-2-oxobutanoyl-CoA
?
show the reaction diagram
-
-
-
-
-
Ethylmalonyl-CoA
(2R)-Methylsuccinyl-CoA
show the reaction diagram
-
reacts about 1000-10000 times moreslowly than the natural substrate
+ a small amount of (2S)-diastereoisomer
-
ethylmalonyl-CoA
methylsuccinyl-CoA
show the reaction diagram
-
reacts about 1000-10000 times moreslowly than the natural substrate
-
-
Methylmalonyl-carba(dethia)-CoA
Succinyl-carby(dethia)-CoA
show the reaction diagram
-
r
-
-
methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
Succinyl-carba(dethia)-CoA
?
show the reaction diagram
-
-
-
-
-
Succinyl-CoA
?
show the reaction diagram
Succinyl-dicarba(dethia)-CoA
?
show the reaction diagram
-
-
-
-
-
additional information
?
-
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
(R)-2-methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
(R)-methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
Succinyl-CoA
?
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-deoxyadenosylcobalamin
adenosylcobalamin
Coalpha(alpha-Benzimidazolyl)-Cobeta-adenosyl-cobamide
-
can serve as coenzyme
Coalpha-(alpha-Purinyl)-Cobeta-adenosylcobamide
-
can serve as coenzyme
Coalpha-Hydroxo-Cobeta-adenosylcobinamide
-
can serve as coenzyme
Coalpha[alpha-(Aden-7-yl)]-Cobeta-adenosyl-cobamide
-
can serve as coenzyme
Cobalamin
cobamide
deoxyadenosylcobinamide GDP
-
-
-
hydroxycobalamin
-
required for Sbm activity
vitamin B12
-
required
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cl-
-
stimulates
NH4+
-
stimulates at 3 mM
PO43-
-
required, maximal activity at 50 mM
Rb+
-
stimulates at 3 mM
SO42-
-
stimulates
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R)-2-Methyl-3-oxopropanoyl-CoA
-
substrate inhibition
1-Carboxyethyl-CoA sulfoxide
-
-
2-(N,N-diethylamino)-diazenolate-2-oxide
-
1 mM, about 50% inhibition of enzyme activity. Similar inhibitory effects of 2-(N,N-diethylamino)-diazenolate-2-oxide in the presence of 25 mM glutathione and dithiothreitol
2-Carboxyethyl-CoA sulfoxide
-
-
5,5'-dithiobis(2-nitrobenzoic acid)
-
-
allylmalonyl-CoA
-
competitive with wild-type, O2-independent suicide inactivation with mutant Y243A via an internal electron transfer from cob(II)alamin to the inhibitor radical
Ca2+
-
at high concentrations
Cyclopropylcarbonyl-CoA carboxylate
-
reversible mixed-type inhibition
ethylmalonyl-CoA
-
competitive with wild-type, O2-dependent suicide inactivation with mutant Y243A
Hg2+
MCM activity is inhibited completely by the addition of 3 mM Hg2+
isobutyryl-CoA
-
R207Q and Y89F/R207Q mutants show conversion of adenosylcobalamin to hydroxycobalamin, indicating inactivation of the enzyme
Li+
-
slight inhibition at 3 mM
Methylenecyclopropylacetyl-CoA
-
reversible mixed-type inhibition
methylmalonyl-CoA
-
reversible mixed-type inhibition
Mg2+
-
at high concentrations
n-Butyryl-CoA
-
R207Q and Y89F/R207Q mutants show conversion of adenosylcobalamin to hydroxycobalamin, indicating inactivation of the enzyme. No changes in wild-type and Y89F-mutant
Na+
-
slight inhibition at 3 mM
NEM
-
-
oxygen
-
600 microM, significant inhibition of enzyme activity
p-chloromercuribenzoate
-
-
p-hydroxymercuribenzoate
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(Cobeta-5'-Deoxyadenosine-5'-yl)-(p-cresolyl)cobamide
-
can serve as coenzyme, Km: 0.000064
adenosylcobalamin
-
required
hydroxocobalamin
-
supplementation of hydroxocobalamin results in a marked increase in the holo-MCM activity in a dose-dependent manner, although the holo-MCM activity does not exceed 30% of the total-MCM activity even if hydroxocobalamin is supplemented at 10 mM
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
0.085 - 1.5
(R)-2-Methyl-3-oxopropanoyl-CoA
0.0279 - 0.364
(R)-2-methylmalonyl-CoA
0.11 - 0.13
(R,S)-methylmalonyl-CoA
0.06 - 0.2
(R/S)-2-methyl-3-oxopropanoyl-CoA
0.132
4-Carboxy-2-oxobutanoyl-CoA
-
-
0.00024 - 0.0086
adenosylcobalamin
0.136
methylmalonyl-carba(dethia)-coenzyme A
0.133 - 0.357
methylmalonyl-CoA
0.025 - 0.3474
succinyl-CoA
2.2
Succinyl-dicarba(dethia)-CoA
-
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.003 - 255
(R)-2-methylmalonyl-CoA
0.012 - 3.95
(R)-methylmalonyl-CoA
150 - 283
(R/S)-2-methyl-3-oxopropanoyl-CoA
0.038 - 48
succinyl-CoA
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0109 - 0.425
allylmalonyl-CoA
0.0153 - 0.467
ethylmalonyl-CoA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000052
-
baby hamster kidney cells grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, lacking hydroxycobalamin, 37C, pH 7.4
0.0002
-
cell extracts preincubated with 5 microM adenosylcobalamin, 37C, pH 7.4
0.06
crude homogenate, at 40C
1.33
-
-
2.2
A4YEG1 and A4YIE3
75C, pH not specified in the publication
10.85
-
after UNO Q-1 purification
21
after 350fold purification, at 40C
67.4
-
-
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
-
active over this pH-range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
75
A4YEG1 and A4YIE3
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
methylmalonyl-CoA mutase and propionyl-CoA carboxylase enzymes are co-expressed in neurons and found in all regions of the central nervous system except in astrocytes and oligodendrocytes. The highest expression in the developing brain is in the periventricular zones of telencephalon, midbrain, and rhombencephalon. The highest expression levels of MCM in adult rat CNS are in neocortex and hippocampal formation, thalamic and hypothalamic nuclei, midbrain nuclei such as the red nucleus and substantia nigra, as well as pons, medulla and the Purkinje and granular layers of cerebellum
Manually annotated by BRENDA team
-
sole carbon and energy source
Manually annotated by BRENDA team
-
sole carbon and energy source
Manually annotated by BRENDA team
-
sole carbon and energy source
Manually annotated by BRENDA team
mandibular lymph node
Manually annotated by BRENDA team
-
peripheral blood lymphocyte
Manually annotated by BRENDA team
oropharyngeal tonsil
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aeropyrum pernix (strain ATCC 700893 / DSM 11879 / JCM 9820 / NBRC 100138 / K1)
Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34)
Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34)
Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34)
Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
78000
-
gel filtration
79000
-
SDS-PAGE
122000 - 124000
-
gel filtration, calculation from sedimentation and diffusion data
144000
-
gel filtration
145000
-
gel filtration
149000
gel filtration
150000
160000
-
Sbm runs at 80000 Da monomer and 160000 Da dimer, non-denaturing PAGE
163000 - 165000
-
sedimentation equilibrium analysis, gel filtration
165000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
heterotetramer
homodimer
monomer
-
1 * 80000, SDS-PAGE, Sbm runs at 80000 Da monomer and 160000 Da dimer
additional information
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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crystal structure at 2 A resolution of methylmalonyl-CoA mutase in complex with coenzyme B12 and with the partial substrate desulfoCoA
-
determination of the structure of substrate-free methylmalonyl-CoA mutase is initiated to provide further insight into the mechanism of radical formation; vapour diffusion at 23C
-
of the enzymatically inactive inhibitor-protein complex with hydroxycob(III)alamin
-
vapour diffusion at 23C, in the presence of succinyl-CoA, 3-carboxylpropyl-CoA or (2R)-carboxylpropyl-CoA
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 50
the enzyme, when incubated at various temperatures for 10 min at pH 7.0, is stable up to 25C, and complete loss of activity is observed at 50C
50
-
15 min, stable below
55
-
t1/2: 3 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
repeated freezing and thawing results in a gradual loss of activity
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10C, 50 mM Tris/HCl buffer, pH 8.0, 1 mM dithiothreitol, about 20-30% loss of activity after 2 days
-
-10C, enzyme from strain 52 W is quite stable, the enzyme from strain St 33 is much less so
-
-10C, stable for at least 3 months
-
-20C, 1.5 mg protein /ml in 50 mM potassium phosphate, pH 7.4, stable for at least 6 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, TSKgel phenyl-Toyopearl column chromatography, TSKgel QAE-Toyopearl column chromatography, Bio-Scale CHT2-1 column chromatography, UNO Q-1 column chromatography, and Superdex 200 gel filtration
expressed in Saccharomyces cerevisiae
-
IMAC column chromatography
-
Ni-NTA agarose resin chromatography
-
Ni-NTA column chromatography, Resource Q column chromatography, and Superdex 200 gel filtration
-
nickel affinity chromatography
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta(DE3)pLysS cells by nickel affinity and anion exchange chromatography
-
to homogeneity
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 (DE3) cells and COS-7 cells
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli BL21(DE3)R3-Rosetta cells
-
expression in COS cells
-
expression in Escherichia coli
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta(DE3)pLysS cells
-
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
-
functional transgenic enzyme expression in enzyme-deficient Mus musculus using a AAV8-CBA-MUT vector
-
mutant enzymes G94V, Y231N, R369H, G623R, H678R and G717V from patients suffering from the mut-form of methylmalonic acidemia, expression in Escherichia coli
-
overexpression in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of the gene is significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO2 fixation
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E392A
-
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
-
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
-
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
-
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
-
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
-
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
-
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
-
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
-
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
H224Q
-
lower turnover than wild-type enzyme
H244A
-
lower turnover than wild-type enzyme
H610A
-
weakened affinity to the cofactor and much lower turnover than wild-type enzyme
H610N
-
weakened affinity to the cofactor and much lower turnover than wild-type enzyme
R207Q
-
active site residue, mutation lowering kcat 10000-fold and increasing KM for methylmalonyl-CoA over 30fold
R207Q/Y86F
-
double mutant designed to mimic active site of isobutyryl-CoA mutase
Y243A
-
40000fold decrease in catalytic efficiency, twofold decrease of cob(II)alamin concentration under steady state turnover conditions, no loss of stereochemical preference for substrate
Y89F
-
active site residue, mutation lowering kcat and increasing KM for methylmalonyl-CoA
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
medicine
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