Cloned (Comment) | Organism |
---|---|
expression of C-terminally His-tagged lipoic acid-bearing domain LBD, amino acid residues 1-84 of E2b | Homo sapiens |
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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Homo sapiens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His-tagged lipoic acid-bearing domain LBD by nickel affinity chromatography | Homo sapiens |
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 Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
- |
Homo sapiens |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at | Homo sapiens |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | bound to E1b component, binding kinetics, wild-type and mutant E1b components | Homo sapiens |