Information on EC 1.2.4.4 - 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)

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

EC NUMBER
COMMENTARY hide
1.2.4.4
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RECOMMENDED NAME
GeneOntology No.
3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] lipoyllysine = [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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oxidative decarboxylation
redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(S)-3-methyl-2-oxopentanoate dehydrogenase (acylating)
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2-oxoisovalerate decarboxylation to isobutanoyl-CoA
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4-methyl-2-oxopentanoate dehydrogenase (acylating)
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NIL
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isoleucine metabolism
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pantothenate biosynthesis
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Valine, leucine and isoleucine degradation
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Propanoate metabolism
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Metabolic pathways
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Biosynthesis of secondary metabolites
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
3-methyl-2-oxobutanoate:[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase]-lipoyllysine 2-oxidoreductase (decarboxylating, acceptor-2-methylpropanoylating)
Contains thiamine diphosphate. It acts not only on 3-methyl-2-oxobutanaoate, but also on 4-methyl-2-oxopentanoate and (S)-3-methyl-2-oxopentanoate, so that it acts on the 2-oxo acids that derive from the action of transaminases on valine, leucine and isoleucine. It is a component of the multienzyme 3-methyl-2-oxobutanoate dehydrogenase complex in which multiple copies of it are bound to a core of molecules of EC 2.3.1.168, dihydrolipoyllysine-residue (2-methylpropanoyl)transferase, which also binds multiple copies of EC 1.8.1.4, dihydrolipoyl dehydrogenase. It does not act on free lipoamide or lipoyllysine, but only on the lipoyllysine residue in EC 2.3.1.168.
CAS REGISTRY NUMBER
COMMENTARY hide
9031-05-4
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9082-72-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
archaebacterium
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
strain DSM 3757
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Manually annotated by BRENDA team
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
plant
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Automatic Mining of ENzyme DAta
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1009208 A+, 1009316 A+, 1009318 A+, 1009434 A+, 1009435 A+, 1720418 A+, 1720422 A+, 1720429 A+, 1720447 A+, 1720489 A+, 1720511 A+, 1720513 A+, 1720514 A+, 1720515 A+, 2243954 A+, 2247234 A+, 2247236 A+, 2247242 A+, 2351118 A+, 2495518 A+, 2760337 A+, 2779760 A+, 2779761 A+, 2779771 A+, 2779783 A+, 2779806 A+, 2779831 A+, 390221 A+, 640578 A+, 640579 A+, 640585 A+, 690337 A+, 721483 A+, 725082 A+, 859030 A+, 1009320 A++, 1009333 A++, 1009340 A++, 1009341 A++, 1009349 A++, 1009354 A++, 1009362 A++, 1009365 A++, 1009368 A++, 1009374 A++, 1009378 A++, 1009389 A++, 1009390 A++, 1009391 A++, 1009394 A++, 1009395 A++, 1009396 A++, 1009397 A++, 1009399 A++, 1009419 A++, 1009420 A++, 1009425 A++, 1158885 A++, 1161709 A++, 1161710 A++, 1161713 A++, 1326110 A++, 1456915 A++, 1720359 A++, 1720419 A++, 1720421 A++, 1720430 A++, 1720431 A++, 1720437 A++, 1720449 A++, 1720451 A++, 1720464 A++, 1720467 A++, 1720476 A++, 1720496 A++, 1720501 A++, 2247194 A++, 2247195 A++, 2247200 A++, 2247201 A++, 2247202 A++, 2247203 A++, 2247207 A++, 2247208 A++, 2247210 A++, 2247213 A++, 2247215 A++, 2247218 A++, 2247219 A++, 2247226 A++, 2247227 A++, 2247228 A++, 2247230 A++, 2247233 A++, 2247235 A++, 2247239 A++, 2247240 A++, 2472662 A++, 2603430 A++, 2603481 A++, 2702061 A++, 2765713 A++, 2779790 A++, 2779792 A++, 2779800 A++, 640592 A++, 662695 A++, 690740 A++, 732945 A++, 1009321 A++, 1009358 A++, 1009403 A++, 1161712 A++, 1720420 A++, 1720428 A++, 1720435 A++, 1720436 A++, 1720440 A++, 1720444 A++, 1720445 A++, 1720452 A++, 1720468 A++, 1720480 A++, 1720483 A++, 1720498 A++, 2213927 A++, 2247192 A++, 2247193 A++, 2247196 A++, 2247209 A++, 2247211 A++, 2247212 A++, 2247214 A++, 2247216 A++, 2247217 A++, 2247224 A++, 2247229 A++, 2247237 A++, 2247244 A++, 2262673 A++, 2779762 A++, 2779777 A++, 2779787 A++, 2779804 A++, 349070 A++, 587061 A++, 640580 A++, 640596 A++, 640597 A++, 640599 A++, 643860 A++, 660663 A++, 1003343 A+++, 1009363 A+++, 1009433 A+++, 1009438 A+++, 710845 A+++, 723788 A+++, 1003378 A++++, 1009323 A++++, 1009327 A++++, 1009344 A++++, 1009351 A++++, 1009356 A++++, 1009357 A++++, 1009360 A++++, 1009364 A++++, 1009369 A++++, 1009370 A++++, 1009371 A++++, 1009375 A++++, 1009376 A++++, 1009380 A++++, 1009381 A++++, 1009382 A++++, 1009383 A++++, 1009386 A++++, 1009387 A++++, 1009398 A++++, 1009402 A++++, 1009404 A++++, 1009405 A++++, 1009415 A++++, 1009416 A++++, 1009423 A++++, 349060 A++++, 349067 A++++, 349068 A++++, 654466 A++++, 712934 A++++
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
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Manually annotated by BRENDA team
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Automatic Mining of ENzyme DAta
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Automatic Mining of ENzyme DAta
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxo-4-methylthiobutanoate + NAD+ + CoA
3-methylthiopropionyl-CoA + NADH + CO2
show the reaction diagram
2-oxo-beta-methylvalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
?
show the reaction diagram
2-oxo-butyrate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
?
show the reaction diagram
2-oxo-gamma-methylthiobutyrate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
?
show the reaction diagram
2-oxo-glutarate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
?
show the reaction diagram
2-oxo-isocaproate + NAD+ + CoA
3-methylbutanoyl-CoA + CO2 + NADH + H+
show the reaction diagram
2-oxo-isocaproate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
?
show the reaction diagram
2-oxobutanoate + NAD+ + CoA
propionyl-CoA + CO2 + NADH
show the reaction diagram
2-oxoglutarate + NAD+ + CoA
3-carboxypropionyl-CoA + NADH + CO2
show the reaction diagram
2-oxoisovalerate + 2,6-dichlorophenol indophenol
? + CO2
show the reaction diagram
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?
2-oxoisovalerate + 2,6-dichlorophenolindophenol + CoA
? + CO2 + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
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?
2-oxoisovalerate + NAD+
? + NADH
show the reaction diagram
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?
2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase] lipoyllysine
?
show the reaction diagram
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?
2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
?
show the reaction diagram
2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
? + CO2
show the reaction diagram
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?
2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-butanoyl-dihydrolipoyllysine + CO2
show the reaction diagram
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overall reaction of the branched-chain alpha-keto acid dehydrogenase multienzyme complex BCKADH
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?
2-oxopentanoate + NAD+ + CoA
butanoyl-CoA + CO2 + NADH
show the reaction diagram
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?
3-methyl-2-oxobutanoate + NAD+ + CoA
2-methylpropanoyl-CoA + CO2 + NADH
show the reaction diagram
3-methyl-2-oxobutanoate + NAD+ + CoA
2-methylpropanoyl-CoA + CO2 + NADH + H+
show the reaction diagram
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?
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
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?
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
3-methyl-2-oxopentanoate + NAD+ + CoA
2-methylbutanoyl-CoA + CO2 + NADH
show the reaction diagram
3-methyl-2-oxopentanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylbutanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
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r
4-methyl-2-oxopentanoate + NAD+ + CoA
3-methylbutanoyl-CoA + CO2 + NADH
show the reaction diagram
4-methyl-2-oxopentanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(3-methylbutanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
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r
acyl-E1b-thiamine diphosphate + lipoyl-[lipoic acid-bearing domain]
2-oxo-acyl-S-lipoyl-[lipoic acid-bearing domain] + E1b-thiamine diphosphate
show the reaction diagram
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reductive acylation of lipoyl-LBD
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?
alpha-keto-beta-methylvaleric acid + NADH
?
show the reaction diagram
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?
alpha-ketoisocaproic acid + NADH
?
show the reaction diagram
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?
alpha-ketoisovaleric acid + NADH
?
show the reaction diagram
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?
E1b-thiamine diphosphate + 2-oxo-acid
E1b-thiamine diphosphate-acyl + CO2
show the reaction diagram
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decarboxylation, His146beta' and His291alpha are involved
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?
pyruvate + NAD+ + CoA
acetyl-CoA + CO2 + NADH
show the reaction diagram
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
2-oxo-isocaproate + NAD+ + CoA
3-methylbutanoyl-CoA + CO2 + NADH + H+
show the reaction diagram
3-methyl-2-oxobutanoate + NAD+ + CoA
2-methylpropanoyl-CoA + CO2 + NADH + H+
show the reaction diagram
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?
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
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?
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
show the reaction diagram
alpha-keto-beta-methylvaleric acid + NADH
?
show the reaction diagram
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?
alpha-ketoisocaproic acid + NADH
?
show the reaction diagram
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?
alpha-ketoisovaleric acid + NADH
?
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thiamine diphosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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required
phosphate
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required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-chloro-4-methylpentanoate
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inhibits component E1, modifies a histidine side chain
2-chloroisocaproate
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2-oxobutanoate
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2-oxoglutarate
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2-Oxopentanoate
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3-methyl-2-oxobutanoate
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substrate inhibition
3-methyl-2-oxopentanoate
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3-methylbutanoyl-CoA
4-(2-Thienyl)-2-oxo-3-butenoate
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4-(3-Thienyl)-2-oxo-3-butenoate
4-methyl-2-oxopentanoate
alpha-Ketoisocaproate
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allosteric inhibition, also by other branched-chain keto acids, although they are less effective as compared to alpha-ketoisocaproate
cinnamylpyruvate
Clofibrate
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D-3-methyl-2-oxopentanoate
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substrate inhibition
Furfurylidenepyruvate
isobutanoyl-CoA
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0.2 mM, 48% inhibition
isopentanoyl-CoA
L-3-Methyl-2-oxopentanoate
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substrate inhibition
microRNA 29a
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total BCKD activity decreases over a 6-day period following a single transfection
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microRNA 29b
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total BCKD activity decreases over a 6-day period following a single transfection, is targeted to the mRNA for the dihydrolipoamide branched chain acyltransferase component of BCKD and prevents translation when bound
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Skeletal muscle factor
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activity from liver, kidney and brains
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additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Activator protein
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a protein in rat liver and kidney mitochondria that reactivates phosphorylated branched-chain complex without dephosphorylation
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alpha-Chloroisocaproate
alpha-Ketoisocaproate
alpha-ketoisovalerate
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activates through release of branched-chain alpha-keto acid dehydrogenase kinase from the BCKD complex
bezafibrate
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i.e. 2-[4-[2-(4-chlorobenzamido)ethyl]phenoxy]-2-methylpropanoic acid. In rats treated with 5, 10, 20 mg/kg bezafibrate mean enzyme actual activity is 1.9, 2.9, and 4.3fold higher than in the control group, respectively. Enzymatic total activity increases by 1.3 and 1.6fold in rats treated with 10 and 20 mg/kg bezafibrate
L-Val
Lipoamide
Skeletal muscle factor
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stimulates enzyme from liver
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thiamine
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thiamine increases the specific activity of the human liver enzyme complex
thiamine diphosphate
tumor necrosis factor-alpha
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0133
2-oxo-4-methylthiobutanoate
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0.028 - 4.38
2-oxo-isovalerate
0.0075 - 0.056
2-oxobutanoate
0.29 - 2.5
2-oxoglutarate
0.00067 - 0.568
3-methyl-2-oxobutanoate
0.01 - 0.25
3-methyl-2-oxopentanoate
0.0062 - 0.2
4-methyl-2-oxopentanoate
0.033 - 0.06
coenzyme A
0.0105 - 0.0172
DL-3-methyl-2-oxopentanoate
0.03 - 0.12
NAD+
0.173 - 2.2
pyruvate
0.00055 - 0.04
thiamine diphosphate
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.195 - 9.4
2-oxo-isovalerate
0.1983 - 2.85
alpha-ketoisovaleric acid
0.197 - 6.38
thiamine diphosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.15
4-(3-Thienyl)-2-oxo-3-butenoate
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0.5
cinnamylpyruvate
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0.0005
Furfurylidenepyruvate
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0.0082 - 0.0136
isovaleryl-CoA
0.0177 - 0.023
NADH
additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0582
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3.13
purified branched-chain alpha-keto acid dehydrogenase multienzyme complex, substrate 2-oxo-isovalerate
5.5
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multienzyme complex
8.8
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multienzyme complex
38
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E1 component expressed in Escherichia coli
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 7.4
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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fetal and maternal
Manually annotated by BRENDA team
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activity increases when plants are placed in the dark, expression is strongly associated with the progression of leaf senescence
Manually annotated by BRENDA team
additional information
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not detected in astrocytes and Bergmann glia cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
plant
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Automatic Mining of ENzyme DAta
PDB
SCOP
CATH
ORGANISM
UNIPROT
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46270
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unlipoylated enzyme, mass spectrometric analysis
46460
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lipoylated enzyme, mass spectrometric analysis
79400
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gel filtration
154000
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E1, heart
160000
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wild-type enzyme and mutant enzymes S293E, S293A, S303A and S293E/S303E, gel filtration
167000
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E1 component, equilibrium sedimentation
172000
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equilibrium sedimentation
190000
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24mer
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E2 component
heterotetramer
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x-ray crystallography
homotetramer
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4 * 19850, gel filtration; 4 * 20000, SDS-PAGE
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
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branched-chain alpha-ketoacid dehydrogenase multienzyme complex BCKDC which is regulated by phosphorylation of component E1b, residue Ser292 of the alpha-domain, the phosphorylated recombinant E1b component shows highly reduced activity compared to the nonphosphorylated one
additional information
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limited proteolytic analysis of E1b component, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
20-25 mg/ml wild-type or recombinant His-tagged E1b component in 50 mM HEPES, pH 7.5, 0.25 M KCl, 0.5 mM PMSF, 1 mM benzamidine, 20 mM DTT, 5% v/v glycerol, vapour diffusion method, 20C, mixing with equal volume of well solution containing 1.4-1.6 M ammonium sulfate, 0.1 M sodium citrate, pH 5.8, 20 mM 2-mercaptoethanol, 4 mM MgCl2 or MnCl2, 4 mM thiamine diphosphate, maximal size after 10 day after microseeding, cryoprotection by well solution with 20% v/v glycerol, X-ray diffraction structure determination and analysis at 1.85-2.25 A resolution
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crystals of E1b grown in the absence and presence of substrates at 22C via the vapor-diffusion method, key tyrosine residue in the E1b active site, functions as a conformational switch to reduce the reactivity of the thiamin diphosphate cofactor, the tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b
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purified wild-type and C-terminally His-tagged recombinant E1b component and mutants, 22C, hanging drop vapour diffusion method, Mg2+ or Mn2+, X-ray diffraction structure determination and analysis at 1.8 A resolution
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crystallization of 4 forms of complex component E1: 1. E1 apoenzyme, 2. E1 holoenzyme, 3. E1 holoenzyme in complex with substrate analogue 4-methylpentanoate, 4. E1 holoenzyme in complex with substrate 4-methyl-2-oxopentanoate, hanging drop vapour diffusion method, 18C, 0.002 ml 10 mg/ml purified recombinant protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, with equal volume of 0.002 ml of reservoir solution containing 0.7 M lithium sulfate, 60 mM sodium citrate, pH 5.6, against 0.4 ml reservoir solution, a few days, X-ray diffraction structure determination and analysis at 1.9-2.4 A resolution
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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20 min, about 85% loss of activity without thiamin diphosphate. 20 min, about 65% loss of activity in presence of 0.2 mM thiamin diphosphate
additional information
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thermal and cold denaturation kinetics of lipoic acid-bearing domain
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
maximum solubilization with minimum inactivation at 44 mM perchlorate carefully timed for 13 min
-
when saturated with thiamin diphosphate, the multienzyme complex is more stable to heat and chymotrypsin inactivation
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, after concentration and dialysis into 50 mM Tris-HCl buffer, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, pH 7.3, multienzyme complex is stable for at least 2 months
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-70C, 10 mM Tris-HC1, pH 8.0, containing 0.01 mM EDTA, 0.2 M NaCI, 0.005% (v/v) 2-mercaptoethanol, and 50% (v/v) glycerol, 2 years, remains active
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-70C, 50 mM potassium phosphate buffer, pH 7.4, 0.1 mM EDTA, 0.2 mM thiamine diphosphate, 0.2 mM phenylmethylsulfonyl fluoride, stable for at least 3 weeks
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-70C, enzyme complex is stable for more than 8 months
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-70C, multienzyme complex is stable for at least 2 years
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0-5C, rapid loss of 20-30% of the activity, then stable for 3-4 months
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0C, glycerol buffer, 20% loss of activity after 24 h
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
branched-chain alpha-keto acid dehydrogenase multienzyme complex BCKADH, consisting of 3 subunits/components E1-E3, from liver mitochondria, isolation of the complex components E1, E2, and E3
by Ni-NTA affinity chromatography
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DEAE-Sepharose CL-6B column chromatography and heparin-Sepharose CL-6B column chromatography
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E1 component, recombinant enzyme
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enzyme complex components E1 and E2 from liver mitochondria