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Literature summary extracted from

  • Li, J.; Wynn, R.M.; Machius, M.; Chuang, J.L.; Karthikeyan, S.; Tomchick, D.R.; Chuang, D.T.
    Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase (2004), J. Biol. Chem., 279, 32968-32978.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.4.4 expression of N-and C-terminally His6-tagged component E1b and mutants, and of C-terminally His-tagged lipoic acid-bearing domain, amino acid residues 1-84 of component E2b, in vitro lipoylation of the latter Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.4.4 purified wild-type and C-terminally His-tagged recombinant E1b component and mutants, 22°C, hanging drop vapour diffusion method, Mg2+ or Mn2+, X-ray diffraction structure determination and analysis at 1.8 A resolution Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
1.2.4.4 D295A site-directed mutagenesis of an alpha-subunit residue of component E1b, increased activity compared to the wild-type Homo sapiens
1.2.4.4 R287A site-directed mutagenesis of an alpha-subunit residue of component E1b, highly increased Km and reduced activity compared to the wild-type Homo sapiens
1.2.4.4 R301A site-directed mutagenesis of an alpha-subunit residue of component E1b, increased Km and reduced activity compared to the wild-type Homo sapiens
1.2.4.4 Y300A site-directed mutagenesis of an alpha-subunit residue of component E1b, increased Km and reduced activity compared to the wild-type Homo sapiens
1.2.4.4 Y300F site-directed mutagenesis of an alpha-subunit residue of component E1b, increased Km and reduced activity compared to the wild-type Homo sapiens
4.1.1.72 D295A of component E1b, 2fold increase in kcat-value Homo sapiens
4.1.1.72 R287A of component E1b, 40fold increase in Km-value for 2-oxoisovalerate Homo sapiens
4.1.1.72 R301A of component E1b, 4fold increase in Km-value for 2-oxoisovalerate Homo sapiens
4.1.1.72 Y300A of component E1b, 6fold increase in Km-value for 2-oxoisovalerate Homo sapiens
4.1.1.72 Y300F of component E1b, 5fold increase in Km-value for 2-oxoisovalerate Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.11.4 additional information binding of thiamin diphosphate cofactor to branched-chain alpha-keto acid decarboxylase/dehydrogenase, which induces a phosphorylation loop conformation change, inhibits the phosphorylation of the protein by the BCKD kinase, no inhibition of phosphorylation of mutant R287A, D295A, Y300F, and R301A E1B components Rattus norvegicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.4.4 additional information
-
additional information kinetics of the overall reaction, thermodynamics and dissociation constants of interaction between component E1b and the lipoic acid-bearing domain of component E2b Homo sapiens
1.2.4.4 0.007
-
thiamine diphosphate recombinant wild-type component E1b, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.017
-
thiamine diphosphate recombinant component E1b mutant D295Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.024
-
thiamine diphosphate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.029
-
thiamine diphosphate recombinant component E1b mutants R301Aalpha and Y300Falpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.04
-
thiamine diphosphate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.05
-
2-oxo-isovalerate recombinant wild-type component E1b, and mutant D295Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.23
-
2-oxo-isovalerate recombinant component E1b mutant R301Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.25
-
2-oxo-isovalerate recombinant component E1b mutant Y300Falpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.34
-
2-oxo-isovalerate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 2.33
-
2-oxo-isovalerate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
4.1.1.72 0.00063
-
thiamine diphosphate wild-type, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.0066
-
thiamine diphosphate wild-type, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.015
-
thiamine diphosphate mutant D295A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.016
-
thiamine diphosphate mutant D295A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.018
-
thiamine diphosphate mutant Y300F, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.026
-
thiamine diphosphate mutant R301A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.026
-
thiamine diphosphate mutant Y300A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.028
-
thiamine diphosphate mutant Y300F, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.029
-
thiamine diphosphate mutant R301A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.04
-
thiamine diphosphate mutant Y300A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.05
-
2-oxoisovalerate mutant D295A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.05
-
2-oxoisovalerate mutant D295A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.05
-
2-oxoisovalerate wild-type, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.05
-
2-oxoisovalerate wild-type, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.14
-
2-oxoisovalerate mutant Y300A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.225
-
2-oxoisovalerate mutant R301A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.252
-
2-oxoisovalerate mutant Y300F, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.255
-
2-oxoisovalerate mutant R301A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.337
-
2-oxoisovalerate mutant Y300A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.39
-
2-oxoisovalerate mutant Y300F, pH 7.5, 30°C, overall reaction Homo sapiens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.2.4.4 Mg2+ required for the binding of cofactor thiamine diphosphate to component E1b, Mn2+ can substitute Homo sapiens
2.7.11.4 Mg2+
-
Rattus norvegicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.4.4 3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine Homo sapiens
-
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
-
?
2.7.11.4 ATP + branched-chain alpha-keto acid decarboxylase/dehydrogenase Rattus norvegicus inactivation of the substrate enzyme ADP + phosphorylated branched-chain alpha-keto acid decarboxylase/dehydrogenase
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.2.4.4 Homo sapiens
-
-
-
2.7.11.4 Rattus norvegicus
-
-
-
4.1.1.72 Homo sapiens
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
1.2.4.4 additional information limited proteolytic analysis of E1b component, overview Homo sapiens

Reaction

EC Number Reaction Comment Organism Reaction ID
1.2.4.4 3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] lipoyllysine = [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2 + 2 H+ substrate recognition and reaction mechanism Homo sapiens
4.1.1.72 (3S)-3-methyl-2-oxopentanoate = 2-methylbutanal + CO2 binding of cofactor thiamine diphosphate to E1b component of enzyme induces a disorder-to-order transition of the conserved phosphorylation loop carrying phosphorylation sites S292 and S302 Homo sapiens

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.2.4.4 additional information
-
decarboxylation activity, 30°C Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.4.4 2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
-
Homo sapiens ? + CO2
-
?
1.2.4.4 3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
-
Homo sapiens [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
-
?
1.2.4.4 additional information the overall reaction proceeds in several steps of the components E1, E2, and E3, overview, conformation of the conserved phosphorylation loop, carrying 2 phosphorylation sites Ser292alpha andSer302alpha, is essential for recognition of lipoylated LBD to initiate E1b-catalyzed reductive acylation, E1b is regulated by reversible phosphorylation through the kinase of the multienzyme complex Homo sapiens ?
-
?
2.7.11.4 ATP + branched-chain alpha-keto acid decarboxylase/dehydrogenase inactivation of the substrate enzyme Rattus norvegicus ADP + phosphorylated branched-chain alpha-keto acid decarboxylase/dehydrogenase
-
?
2.7.11.4 ATP + branched-chain alpha-keto acid decarboxylase/dehydrogenase recombinant human wild-type and mutant substrate proteins, overview, phosphorylation at Ser301 and Ser302 in the phosphorylation loop of decarboxylase E1b component of the large branched-chain alpha-keto acid dehydrogenase complex, loop structure, overview Rattus norvegicus ADP + phosphorylated branched-chain alpha-keto acid decarboxylase/dehydrogenase
-
?
4.1.1.72 2-oxoisovalerate
-
Homo sapiens 2-methylpropanal + CO2
-
?

Synonyms

EC Number Synonyms Comment Organism
1.2.4.4 BCKD
-
Homo sapiens
1.2.4.4 branched-chain alpha-keto acid decarboxylase/dehydrogenase
-
Homo sapiens
2.7.11.4 BCKD kinase
-
Rattus norvegicus
2.7.11.4 branched-chain alpha-keto acid decarboxylase/dehydrogenase kinase
-
Rattus norvegicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.11.4 22
-
assay at room temperature Rattus norvegicus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.4.4 0.195
-
2-oxo-isovalerate recombinant wild-type component E1b, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.197
-
thiamine diphosphate recombinant wild-type component E1b, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.31
-
thiamine diphosphate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.36
-
2-oxo-isovalerate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.38
-
2-oxo-isovalerate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.39
-
thiamine diphosphate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.41
-
2-oxo-isovalerate recombinant component E1b mutant R301Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.42
-
thiamine diphosphate recombinant component E1b mutants R301Aalpha and D295Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.485
-
2-oxo-isovalerate recombinant component E1b mutant D295Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.53
-
thiamine diphosphate recombinant component E1b mutant Y300Falpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.57
-
2-oxo-isovalerate recombinant component E1b mutant Y300Falpha, pH 7.5, 22°C Homo sapiens
4.1.1.72 0.19
-
thiamine diphosphate wild-type, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.19
-
2-oxoisovalerate wild-type, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.19
-
thiamine diphosphate wild-type, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.19
-
2-oxoisovalerate wild-type, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.31
-
thiamine diphosphate mutant Y300A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.31
-
thiamine diphosphate mutant Y300A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.38
-
2-oxoisovalerate mutant Y300A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.38
-
2-oxoisovalerate mutant Y300A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.41
-
thiamine diphosphate mutant R301A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.41
-
2-oxoisovalerate mutant R301A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.41
-
thiamine diphosphate mutant R301A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.41
-
2-oxoisovalerate mutant R301A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.42
-
thiamine diphosphate mutant D295A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.42
-
thiamine diphosphate mutant D295A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.48
-
2-oxoisovalerate mutant D295A, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.48
-
2-oxoisovalerate mutant D295A, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.53
-
thiamine diphosphate mutant Y300F, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.53
-
thiamine diphosphate mutant Y300F, pH 7.5, 30°C, overall reaction Homo sapiens
4.1.1.72 0.57
-
2-oxoisovalerate mutant Y300F, pH 7.5, 30°C, E1b-catalyzed decarboxylation reaction Homo sapiens
4.1.1.72 0.57
-
2-oxoisovalerate mutant Y300F, pH 7.5, 30°C, overall reaction Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.4.4 7.5
-
assay at Homo sapiens
2.7.11.4 7.4
-
assay at Rattus norvegicus

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.4.4 thiamine diphosphate bound to component E1, heterotetrameric cofactor binding fold, cofactor binding prevents phosphorylation of E1b and inactivates it by inducing a disorder-to-order transition of the conserved phosphorylation loop carrying 2 phosphorylation sites Ser292alpha andSer302alpha, cross-talk between thiamine diphosphate and the phosphorylation loop conformation as a feed-forward switch for th complex reaction Homo sapiens
2.7.11.4 ATP
-
Rattus norvegicus
4.1.1.72 thiamine diphosphate binds to E1b component of enzyme complex Homo sapiens