Branched chain aminotransferase

Branched-chain amino acid aminotransferase (BCAT), also known as branched-chain amino acid transaminase, is an aminotransferase enzyme (EC 2.6.1.42) which acts upon branched-chain amino acids (BCAAs). It is encoded by the BCAT2 gene in humans. The BCAT enzyme catalyzes the conversion of BCAAs and α-ketoglutarate into branched chain α-keto acids and glutamate.(See Template:Leucine metabolism in humans – this diagram does not include the pathway for β-leucine synthesis via leucine 2,3-aminomutase) Branched-chain amino acid aminotransferase (BCAT), also known as branched-chain amino acid transaminase, is an aminotransferase enzyme (EC 2.6.1.42) which acts upon branched-chain amino acids (BCAAs). It is encoded by the BCAT2 gene in humans. The BCAT enzyme catalyzes the conversion of BCAAs and α-ketoglutarate into branched chain α-keto acids and glutamate. The structure to the right of branched chain amino acid aminotransferase was found using X-ray diffraction with a resolution of 2.20 Å. The branched-chain amino acid aminotransferase found in this image was isolated from mycobacteria. This protein is made up of two identical polypeptide chains. The protein is a total of 372 residues. As can be seen in the image, the protein is made of helices and beta sheets. The biological function of branched-chain amino acid aminotransferases is to catalyse the synthesis or degradation of the branched chain amino acids leucine, isoleucine, and valine. In humans, branched chain amino acids are essential and are degraded by BCATs. In humans, BCATs are homodimers composed of two domains, a small subunit (residues 1-170) and a large subunit (residues 182-365). These subunits are connected by a short, looping connecting region (residues 171-181). Both subunits consist of four alpha-helices and a beta-pleated sheet. Structural studies of human branched-chain amino acid aminotransferases (hBCAT) revealed that the peptide bonds in both isoforms are all trans except for the bond between residues Gly338-Pro339. The active site of the enzyme lies in the interface between the two domains. Like other transaminase enzymes (as well as many enzymes of other classes), BCATs require the cofactor pyridoxal-5'-phosphate(PLP) for activity. PLP has been found to change the conformation of aminotransferase enzymes, locking the conformation of the enzyme via a Schiff base (imine) linkage in a reaction between a lysine residue of the enzyme and the carbonyl group of the cofactor. This conformational change allows the substrates to bind to the active site pocket of the enzymes. In addition to the Schiff base linkage, PLP is anchored to the active site of the enzyme via hydrogen bonding at the Tyr 207 and Glu237 residues. In addition, the phosphate oxygen atoms on the PLP molecule interact with the Arg99, Val269, Val270, and Thr310 residues. Mammalian BCATs show a unique structural CXXC motif (Cys315 and Cys318) sensitive to oxidizing agents and modulated through S-nitrosation, a post-translational modification that regulates cell signaling. Modification of these two cysteine residues via oxidation (in vivo/vitro) or titration (in vitro) has been found to inhibit enzyme activity, indicating that the CXXC motif is crucial to optimal protein folding and function. The sensitivity of both isoenzymes to oxidation make them potential biomarkers for the redox environment within the cell. Although the CXXC motif is present only in mammalian BCATs, the surrounding amino acid residues were found to be highly conserved in both prokaryotic and eukaryotic cells. Conway, Yeenawar et al. found that the mammalian active site contains three surfaces: surface A (Phe75, Tyr207 and Thr240), surface B (Phe30, Tyr141, and Ala314), and surface C (Tyr70, Leu153 and Val155, located on the opposite domain) that bind to the substrate in a Van der Waals-type interaction with the branched side chains of the amino acid substrates. BCATs in mammals catalyze the first step in branched-chain amino acid metabolism, a reversible transamination followed by the oxidative decarboxylation of the transamination products α-ketoisocaproate, α-keto-β-methylvalerate, and α-ketoisovalerate to isovaleryl-CoA, 3-methylbutyryl-CoA, and isobutyryl-CoA, respectively. This reaction regulates metabolism of amino acids and is a crucial step in nitrogen shuttling throughout the whole body. Branched-chain amino acids (BCAA) are ubiquitous in many organisms, comprising 35% of all proteins and 40% of the amino acids required in all mammals. Mammalian BCATs come in two isoforms: cytosolic (BCATc) and mitochondrial (BCATm). The isoforms share 58% homology, but vary in location and catalytic efficiency. Cytosolic branched-chain amino acid aminotransferases are the less common of the two isoforms, found in the cytoplasm of mammalian cells almost exclusively throughout the nervous system. Although BCATc are expressed only in a few adult tissues, they are expressed at a high level during embryogenesis. The cytosolic isoform has a higher turnover rate, approximately 2-5 times faster than the mitochondrial isoform. BCATc has been found to be more stable than BCATm, with evidence suggesting 2 sulfide bonds. The cytosolic isozyme demonstrates no loss in activity upon titration of one thiol group hBCATc demonstrates a lower redox potential (approximately 30 mV) than hBCATm. Mitochondrial branched-chain amino acid aminotransferases are the more ubiquitous of the two isoforms, present in all tissues in the mitochondria of the cell. Pancreatic acinar tissue has been found to carry the highest levels of BCATm in the body In addition, two homologs to normal BCATm have been found. One homolog is found in placental tissue, and the other co-represses thyroid hormone nuclear receptors. BCATm is more sensitive to the redox environment of the cell, and can be inhibited by nickel ions even if the environment is reducing. BCATm has been found to form no disulfide bonds, and titration of two -SH groups with 5,5'- dithiobis(2-nitrobenzoic acid) eliminates enzyme activity completely in the case of the BCATm isozyme. Plant BCATs have also been identified, but vary between species in terms of number and sequence. In studies ofArabidopsis thaliana (thale cress), six BCAT isoforms have been identified that share between 47.5-84.1% homology with each other. These isoforms also share around 30% sequence homology to the human and yeast (Saccharomyces cerevisiae) isoforms. BCAT1 is located in the mitochondria, BCAT2, 3, and 5 are located in chloroplasts, and BCAT4 and 6 are located in the cytoplasm of A. thaliana. However, studies of BCATs in Solanum tuberosum (potato) revealed two isoforms that are 683 (BCAT1) and 746 (BCAT2) bp long located primarily in chloroplasts.