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Literature summary for 1.2.4.4 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)

Cloned (Comment) Organism
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)

Crystallization (Comment) Organism
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

Protein Variants Comment Organism
D295A site-directed mutagenesis of an alpha-subunit residue of component E1b, increased activity compared to the wild-type Homo sapiens
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
R301A site-directed mutagenesis of an alpha-subunit residue of component E1b, increased Km and reduced activity compared to the wild-type Homo sapiens
Y300A site-directed mutagenesis of an alpha-subunit residue of component E1b, increased Km and reduced activity compared to the wild-type Homo sapiens
Y300F site-directed mutagenesis of an alpha-subunit residue of component E1b, increased Km and reduced activity compared to the wild-type Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
0.007
-
thiamine diphosphate recombinant wild-type component E1b, pH 7.5, 22°C Homo sapiens
0.017
-
thiamine diphosphate recombinant component E1b mutant D295Aalpha, pH 7.5, 22°C Homo sapiens
0.024
-
thiamine diphosphate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
0.029
-
thiamine diphosphate recombinant component E1b mutants R301Aalpha and Y300Falpha, pH 7.5, 22°C Homo sapiens
0.04
-
thiamine diphosphate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
0.05
-
2-oxo-isovalerate recombinant wild-type component E1b, and mutant D295Aalpha, pH 7.5, 22°C Homo sapiens
0.23
-
2-oxo-isovalerate recombinant component E1b mutant R301Aalpha, pH 7.5, 22°C Homo sapiens
0.25
-
2-oxo-isovalerate recombinant component E1b mutant Y300Falpha, pH 7.5, 22°C Homo sapiens
0.34
-
2-oxo-isovalerate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
2.33
-
2-oxo-isovalerate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required for the binding of cofactor thiamine diphosphate to component E1b, Mn2+ can substitute Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine Homo sapiens
-
[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens
-
-
-

Posttranslational Modification

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

Reaction

Reaction Comment Organism Reaction ID
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

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
-
Homo sapiens ? + CO2
-
?
3-methyl-2-oxobutanoate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine
-
Homo sapiens [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine + CO2
-
?
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 ?
-
?

Synonyms

Synonyms Comment Organism
BCKD
-
Homo sapiens
branched-chain alpha-keto acid decarboxylase/dehydrogenase
-
Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.195
-
2-oxo-isovalerate recombinant wild-type component E1b, pH 7.5, 22°C Homo sapiens
0.197
-
thiamine diphosphate recombinant wild-type component E1b, pH 7.5, 22°C Homo sapiens
0.31
-
thiamine diphosphate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
0.36
-
2-oxo-isovalerate recombinant component E1b mutant Y300Aalpha, pH 7.5, 22°C Homo sapiens
0.38
-
2-oxo-isovalerate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
0.39
-
thiamine diphosphate recombinant component E1b mutant R287Aalpha, pH 7.5, 22°C Homo sapiens
0.41
-
2-oxo-isovalerate recombinant component E1b mutant R301Aalpha, pH 7.5, 22°C Homo sapiens
0.42
-
thiamine diphosphate recombinant component E1b mutants R301Aalpha and D295Aalpha, pH 7.5, 22°C Homo sapiens
0.485
-
2-oxo-isovalerate recombinant component E1b mutant D295Aalpha, pH 7.5, 22°C Homo sapiens
0.53
-
thiamine diphosphate recombinant component E1b mutant Y300Falpha, pH 7.5, 22°C Homo sapiens
0.57
-
2-oxo-isovalerate recombinant component E1b mutant Y300Falpha, pH 7.5, 22°C Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
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