Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme mutant C318A, hanging drop vapor diffusion method, mixing of 0.001 ml of 5 mg/ml protein in potassium phosphate, pH 7.5, with 0.001 ml of reservoir solution containing 200 mM magnesium acetate tetrahydrate, 100 mM sodium cacodylate trihydrate, pH 6.5, and 20% w/v PEG 8000, and equilibration against 0.5 ml of reservoir solution, 20°C, for oxidation of the C318A mutant, the protein crystals obtained are incubated in crystallization buffer with the addition of 1% hydrogen peroxide before applying the cryoprotectant, X-ray diffraction structure determination and analysis at 3.25 A resolution | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
C315A | site-directed mutagenesis, mutation of the redox sensor (Cys315) results in a significant loss of activity compared to wild-type | Homo sapiens |
C318A | site-directed mutagenesis, the mutant shows unaltered activity compared to wild-type, structure analysis | Homo sapiens |
C318A/C315CSD | the overall structure of the C318A/C315CSD variant overlays very well with that of the C318A mutant and the oxidized form of wild-type hBCATm, with a few exceptions in the interdomain loop (residues 171-181) and the N-terminal loop (residues 15-32) | Homo sapiens |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
mitochondrion | - |
Homo sapiens | 5739 | - |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-leucine + 2-oxoglutarate | Homo sapiens | - |
4-methyl-2-oxopentanoate + L-glutamate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | O15382 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, anion exchange chromatography, and dialysis | Homo sapiens |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
brain | - |
Homo sapiens | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-leucine + 2-oxoglutarate | - |
Homo sapiens | 4-methyl-2-oxopentanoate + L-glutamate | - |
r |
Subunits | Comment | Organism |
---|---|---|
homodimer | - |
Homo sapiens |
Synonyms | Comment | Organism |
---|---|---|
Bcat2 | - |
Homo sapiens |
branched-chain aminotransferase | - |
Homo sapiens |
hBCAT | - |
Homo sapiens |
hBCATm | - |
Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | PLP, dependent on, the reaction is accompanied by the interconversion of the cofactor pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate. PLP is linked to Lys202 by a Schiff base | Homo sapiens |
General Information | Comment | Organism |
---|---|---|
malfunction | mutation of the redox sensor (Cys315) results in a significant loss of activity, with no loss of activity reported on the mutation of the resolving cysteine (Cys318), which allows the reversible formation of a disulfide bond between Cys315 and Cys318 | Homo sapiens |
additional information | active site structure | Homo sapiens |
physiological function | human branched-chain aminotransferase (hBCAT) catalyzes the transamination of the branched-chain amino acids leucine, valine and isoleucine and 2-oxoglutarate to their respective 2-oxo acids and glutamate. hBCAT activity is regulated by a CXXC center located approx. 10 A from the active site. This redox-active center facilitates recycling between the reduced and oxidized states, representing hBCAT in its active and inactive forms, respectively. The structure reveals the modified CXXC center in a conformation similar to that in the oxidized wild type, supporting the notion that its regulatory mechanism depends on switching the Cys315 side chain between active and inactive conformations. The enzyme plays a significant role in amino-acid metabolism and whole-body nitrogen shuttling, in particular with respect to the de novo synthesis of the neurotransmitter glutamate in the brain | Homo sapiens |