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Literature summary extracted from

  • Wynn, R.M.; Machius, M.; Chuang, J.L.; Li, J.; Tomchick, D.R.; Chuang, D.T.
    Roles of His291-alpha and His146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase: refined phosphorylation loop structure in the active site (2003), J. Biol. Chem., 278, 43402-43410.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.4.4 expression of C-terminally His-tagged lipoic acid-bearing domain LBD, amino acid residues 1-84 of E2b Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.4.4 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, 20°C, 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 Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
1.2.4.4 H146A site-directed mutagenesis, mutation of a beta'-subunit residue, no reductive acylation activity, low decarboxylation activity Homo sapiens
1.2.4.4 H147N site-directed mutagenesis, mutation of a beta'-subunit residue, no reductive acylation activity, low decarboxylation activity Homo sapiens
1.2.4.4 H291A site-directed mutagenesis, mutation of a alpha-subunit residue, highly reduced activity compared to the wild-type E1b component Homo sapiens
1.2.4.4 H291N site-directed mutagenesis, mutation of a alpha-subunit residue, highly reduced activity compared to the wild-type E1b component Homo sapiens
1.2.4.4 H291Q site-directed mutagenesis, mutation of a alpha-subunit residue, highly reduced activity compared to the wild-type E1b component Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.4.4 additional information
-
 additional information binding between E1b, wild-type and mutant, and E2b/lipoic acid-bearing domain components and thermodynamics, overall reaction kinetics, overview Homo sapiens
1.2.4.4 0.00055
-
 thiamine diphosphate recombinant wild-type E1b component, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.0014
-
 thiamine diphosphate recombinant E1b component mutant H291Nalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.009
-
 thiamine diphosphate recombinant E1b component mutant H291Qalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.024
-
 thiamine diphosphate recombinant E1b component mutant H291Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.059
-
 2-oxo-isovalerate recombinant wild-type E1b component, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.118
-
 2-oxo-isovalerate recombinant E1b component mutant H291Nalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.203
-
 2-oxo-isovalerate recombinant E1b component mutant H291Qalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.206
-
 2-oxo-isovalerate recombinant E1b component mutant H291Aalpha, pH 7.5, 22°C Homo sapiens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.2.4.4 Mg2+
-
 Homo sapiens

Organism

EC Number Organism UniProt Comment Textmining
1.2.4.4 Homo sapiens
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.4.4 recombinant C-terminally His-tagged lipoic acid-bearing domain LBD by nickel affinity chromatography 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+ residues His146beta' and His291alpha of E1b component are essential for catalysis Homo sapiens
a

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
-
-
 Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.4.4 2-oxoisovalerate + 2,6-dichlorophenol indophenol
-
 Homo sapiens ? + CO2
-
 ?
1.2.4.4 2-oxoisovalerate + [dihydrolipoyllysine-residue(2-methylpropanoyl)transferase]-lipoyllysine overall reaction of the branched-chain alpha-keto acid dehydrogenase multienzyme complex BCKADH Homo sapiens [dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-butanoyl-dihydrolipoyllysine + CO2
-
 ?
1.2.4.4 acyl-E1b-thiamine diphosphate + lipoyl-[lipoic acid-bearing domain] reductive acylation of lipoyl-LBD Homo sapiens 2-oxo-acyl-S-lipoyl-[lipoic acid-bearing domain] + E1b-thiamine diphosphate
-
 ?
1.2.4.4 E1b-thiamine diphosphate + 2-oxo-acid decarboxylation, His146beta' and His291alpha are involved Homo sapiens E1b-thiamine diphosphate-acyl + CO2
-
 ?
1.2.4.4 additional information overall reaction Homo sapiens ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.2.4.4 More the lipoic acid-bearing domain LBD, amino acid residues 1-84, is part of the E2b complex component, which is part of the the branched-chain alpha-ketoacid dehydrogenase multienzyme complex BCKD Homo sapiens

Synonyms

EC Number Synonyms Comment Organism
1.2.4.4 alpha-ketoacid dehydrogenase i.e. E1b enzyme complex component Homo sapiens
1.2.4.4 dihydrolipoyl transacylase i.e. E2b enzyme complex component Homo sapiens
1.2.4.4 More E1b and E2b are components of the branched-chain alpha-ketoacid dehydrogenase multienzyme complex BCKD Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.4.4 22
-
 assay at Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.4.4 0.41
-
 thiamine diphosphate recombinant E1b component mutant H291Qalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.49
-
 2-oxo-isovalerate recombinant E1b component mutant H291Qalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.58
-
 2-oxo-isovalerate recombinant E1b component mutant H291Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.6
-
 thiamine diphosphate recombinant E1b component mutant H291Nalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.67
-
 2-oxo-isovalerate recombinant E1b component mutant H291Nalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 0.73
-
 thiamine diphosphate recombinant E1b component mutant H291Aalpha, pH 7.5, 22°C Homo sapiens
1.2.4.4 6.38
-
 thiamine diphosphate recombinant wild-type E1b component, pH 7.5, 22°C Homo sapiens
1.2.4.4 8.65
-
 2-oxo-isovalerate recombinant wild-type E1b component, pH 7.5, 22°C Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.4.4 7.5
-
 assay at Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.4.4 thiamine diphosphate bound to E1b component, binding kinetics, wild-type and mutant E1b components Homo sapiens