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Literature summary for 1.2.4.4 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)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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

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

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
0.00055
-
thiamine diphosphate recombinant wild-type E1b component, pH 7.5, 22°C Homo sapiens
0.0014
-
thiamine diphosphate recombinant E1b component mutant H291Nalpha, pH 7.5, 22°C Homo sapiens
0.009
-
thiamine diphosphate recombinant E1b component mutant H291Qalpha, pH 7.5, 22°C Homo sapiens
0.024
-
thiamine diphosphate recombinant E1b component mutant H291Aalpha, pH 7.5, 22°C Homo sapiens
0.059
-
2-oxo-isovalerate recombinant wild-type E1b component, pH 7.5, 22°C Homo sapiens
0.118
-
2-oxo-isovalerate recombinant E1b component mutant H291Nalpha, pH 7.5, 22°C Homo sapiens
0.203
-
2-oxo-isovalerate recombinant E1b component mutant H291Qalpha, pH 7.5, 22°C Homo sapiens
0.206
-
2-oxo-isovalerate recombinant E1b component mutant H291Aalpha, pH 7.5, 22°C Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
Homo sapiens

Organism

Organism UniProt Comment Textmining
Homo sapiens
-
-
-

Purification (Commentary)

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

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
-
Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-oxoisovalerate + 2,6-dichlorophenol indophenol
-
Homo sapiens ? + CO2
-
?
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
-
?
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
-
?
E1b-thiamine diphosphate + 2-oxo-acid decarboxylation, His146beta' and His291alpha are involved Homo sapiens E1b-thiamine diphosphate-acyl + CO2
-
?
additional information overall reaction Homo sapiens ?
-
?

Subunits

Subunits Comment Organism
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

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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