Information on EC 5.4.99.4 - 2-Methyleneglutarate mutase

Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Mark a special word or phrase in this record:
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Eubacterium barkeri

EC NUMBER
COMMENTARY
5.4.99.4
-
RECOMMENDED NAME
GeneOntology No.
2-Methyleneglutarate mutase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
the enzyme catalyzes the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon
-
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
mechanism
-
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
rotation of the exo-methylene group of 2-methyleneglutarate
-
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
mechanism; the steric course of the reaction is such that the acrylyl residue migrates from the beta-carbon to the alpha-carbon of the propionate with inversion of configuration at the alpha-carbon; unimolecular free radical rearrangement via 3-butenyl and cyclopropylmethyl intermediates is a reasonable mechanism for the rearrangement step of the enzyme
-
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
mechanism via protein-bound free radicals
-
2-Methyleneglutarate = 2-methylene-3-methylsuccinate
show the reaction diagram
formation of an unusual oxygen-sensitive Co(II) species during catalysis
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
group transfer
-
-
intramolecular
-
isomerization
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Nicotinate and nicotinamide metabolism
-
nicotinate degradation III
-
SYSTEMATIC NAME
IUBMB Comments
2-methyleneglutarate carboxy-methylenemethylmutase
Requires a cobamide coenzyme.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
alpha-Methyleneglutarate mutase
-
-
-
-
alpha-MG mutase
-
-
-
-
Mutase, 2-methyleneglutarate
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9059-10-3
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-3-[methyl-H3]methylitaconate
2-methyleneglutarate
show the reaction diagram
-
-
-
r
(R,Z)-3-methyl[2'-H1]itaconate
2-methyleneglutarate
show the reaction diagram
-
-
-
r
(R,Z)-3-[methyl-H3]methyl[2'-H1]itaconate
2-methyleneglutarate
show the reaction diagram
-
-
-
r
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
-
-
?
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
-
i.e methylitaconate
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
r
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
r
-
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
r
i.e methylitaconate
-
2-Methyleneglutarate
2-Methylene-3-methylsuccinate
show the reaction diagram
-
r, the equilibrium favours the formation of 2-methyleneglutarate
i.e methylitaconate
-
2-methyleneglutarate
(R)-3-methylitaconate
show the reaction diagram
-
-
-
r
2-methylene[4-H2]glutarate
(R)-3-methylitaconate
show the reaction diagram
-
-
-
r
additional information
?
-
-
inducible enzyme, participates in the anaerobic conversion of nicotinic acid to equimolar amounts of propionate
-
-
-
additional information
?
-
-
enzyme is induced by growth on nicotinic acid and is not detectable in cells grown on glucose
-
-
-
additional information
?
-
-
carbon-skeleton rearrangement mechanism. Adenosylcobalamin acts as a carrier for the hydrogen atom that is abstracted from alpha-methyleneglutarate and replaced by the acrylyl group of 2-methyleneglutarate
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
inducible enzyme, participates in the anaerobic conversion of nicotinic acid to equimolar amounts of propionate
-
-
-
additional information
?
-
-
enzyme is induced by growth on nicotinic acid and is not detectable in cells grown on glucose
-
-
-
additional information
?
-
-
carbon-skeleton rearrangement mechanism. Adenosylcobalamin acts as a carrier for the hydrogen atom that is abstracted from alpha-methyleneglutarate and replaced by the acrylyl group of 2-methyleneglutarate
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
adenosylcobalamin
-
-
cob(II)alamin
-
-
cobamide
-
required
cobamide
-
e.g. alpha-(5,6-dimethylbenzimidazolyl)cobamide coenzyme, Km: 0.000073 mM, alpha-(benzimidazolyl)-cobamide coenzyme, Km: 0.0003 mM, or alpha-(adenyl)-cobamide coenzyme, Km: 0.00125 mM. The most effective coenzyme is alpha(5,6-dimethylbenzimidazolyl)-cobamide coenzyme; required
cobamide
-
acts as a carrier for the hydrogen atom that is abstracted from alpha-methyleneglutarate. The enzyme contains 4 cobalamin molecules per tetramer. The cobalamins consist of adenosylcobalamin and cobalamin(II). Enzyme containing adenosylcobalamin is the active form of the enzyme but the enzyme containing cobalamin(II) is stable to oxygen and is an inactive form of the enzyme
cobamide
-
dependent on
cobamide
-
the enzyme contains adenosylcobalamin and varying amounts of oxygen-stable cob(II)alamin. The content of total cobalamin is 2-4 mol/ml enzyme, the content of cob(II)alamin is 6-11% of the total cobalamin. The oxygen-stable cob(II)alamin is not involved in catalysis
vitamin B12
-
dependent on
vitamin B12
-
contains 2.1 mol of this coenzyme per homotetramer
vitamin B12
-
dependent on; during catalysis the Co-C bond of the coenzyme is cleaved
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(Z)-glutaconate
-
mechanism-based inactivation. Inhibitor induces homolysis of the Co-C bond of coenzyme B12 to afford cob(II)alamin and the 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical adds to the double bond of (Z)-glutaconate to afford a radical adduct and subsequent formation of aquocobalamin
1-Methyl-1,2-cis-cyclopropanedicarboxylate
-
-
1-Methyl-1,2-cis-cyclopropanedicarboxylate
-
noncompetitive, slight inhibitor
1-Methyl-1,2-trans-cyclopropanedicarboxylate
-
-
1-Methyl-1,2-trans-cyclopropanedicarboxylate
-
competitive
1-methylcyclopropane-(1R,2R)-dicarboxylate
-
weak noncompetitive
-
buta-1,3-diene-2,3-dicarboxylate
-
mechanism-based inactivation. Inhibitor induces homolysis of the Co-C bond of coenzyme B12 to afford cob(II)alamin and the 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical adds to a double bond of buta-1,3-diene-2,3-dicarboxylate to afford a relatively stable radical adduct in the active site of the enzyme
citrate
-
slight
glutaconate
-
noncompetitive
iodoacetamide
-
alkylated enzyme
iodoacetamide
-
-
Itaconate
-
competitive
L-Malate
-
competitive
Maleate
-
competitive
-
mesaconate
-
competitive
p-Chloromercuriphenylsulfonic acid
-
-
succinate
-
competitive
additional information
-
the enzyme is inactivated by light of the wavelength, lambda = 620 nm. Reactivation of up to 50% of the activity is achieved by incubation with coenzyme B12 and dithiothreitol, the substrates 2-methgyleneglutarate or 3-methylitaconate specifically protect the enzyme from inactivation by visible light
-
additional information
-
acrylate and 2-methylpent-2-enedioate are noninhibitory
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
sulfhydryl groups
-
the enzyme is a sulfhydryl protein
sulfhydryl groups
-
contains active thiol groups
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.4
-
(R)-3-methylitaconate
-
pH 7.4
0.38
-
(R)-3-[methyl-H3]methylitaconate
-
pH 7.4
3.1
-
2-Methyleneglutarate
-
pH 7.4
4
-
2-Methyleneglutarate
-
-
7.1
-
2-Methyleneglutarate
-
-
2.3
-
2-methylene[4-H2]glutarate
-
pH 7.4
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
18
-
(R)-3-methylitaconate
-
pH 7.4
1.1
-
(R)-3-[methyl-H3]methylitaconate
-
pH 7.4
17
-
(R,Z)-3-methyl[2'-H1]itaconate
-
pH 7.4
1.4
-
(R,Z)-3-[methyl-H3]methyl[2'-H1]itaconate
-
pH 7.4
30
-
2-Methyleneglutarate
-
pH 7.4
2.3
-
2-methylene[4-H2]glutarate
-
pH 7.4
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
13
-
1-methylcyclopropane-(1R,2R)-dicarboxylate
-
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.51
-
-
preparation which is heavily contaminated with methylitaconate isomerase
additional information
-
-
-
additional information
-
-
continuous spectrophotometric assay
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
8
-
sharp decrease in activity below pH 7.5 and above pH 8
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
170000
-
-
sucrose density gradient centrifugation
290000
-
-
gel filtration
300000
-
-
gel filtration, sucrose density gradient centrifugation, nondenaturing PAGE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
tetramer
-
4 * 70000, SDS-PAGE
tetramer
-
alpha4, 4 * 67000
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
enzyme rapidly decays, when stored at low protein concentrations, 1 mg/ml. Complete loss of activity after 3 days at 0C and 38% loss of activity after 6 days at -80C
-
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
enzyme containing cobalamin(II) is stable to oxygen and is an inactive form of the enzyme
-
3526
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-80C, protein concentration 1 mg/ml, 38% loss of activity after 6 days
-
-80C, protein concentration 10 mg/ml, about 10% loss of activity after 6 months
-
0C, protein concentration 1 mg/ml, complete loss of activity after 3 days
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
large scale
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
overexpression in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D483N
-
0.06%of wild-type activity
H464Q
-
35% of wild-type activity
H485Q
-
less than 0.03% of wild-type activity
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation affords an inactive enzyme which contains 40-50% of the initially bound cobalamin
-