Yennawar N, Dunbar J, Conway M, Hutson S, Farber G
Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA 16802, USA.
Acta Crystallogr D Biol Crystallogr. 2001 Apr;57(Pt 4):506-15. doi: 10.1107/s0907444901001925.
X-ray crystal structures of three forms of human mitochondrial branched-chain aminotransferase (BCAT) were solved by molecular-replacement methods, using Escherichia coli BCAT as the search model. The enzyme is a homodimer and the polypeptide chain of each monomer has two domains. The small domain is composed of residues 1--175 and the large domain is composed of residues 176--365. The active site is close to the dimer interface. The 4'-aldehyde of the PLP cofactor is covalently linked to the epsilon-amino group of the active-site lysine, Lys202, via a Schiff-base linkage in two of the structures. In the third structure, the enzyme is irreversibly inactivated by Tris. The overall fold of the dimer in human mitochondrial BCAT is similar to the structure of two bacterial enzymes, E. coli BCAT and D-amino acid aminotransferase (D-AAT). The residues lining the putative substrate-binding pocket of human BCAT and D-AAT are completely rearranged to allow catalysis with substrates of opposite stereochemistry. In the case of human mitochondrial branched-chain aminotransferase, a hydrogen-bond interaction between the guanidinium group of Arg143 in the first monomer with the side-chain hydroxyl of Tyr70 in the second monomer is important in the formation of the substrate-binding pocket.
采用分子置换法，以大肠杆菌支链氨基酸转氨酶（BCAT）作为搜索模型，解析了三种形式的人线粒体支链氨基酸转氨酶（BCAT）的X射线晶体结构。该酶为同型二聚体，每个单体的多肽链有两个结构域。小结构域由第1至175位残基组成，大结构域由第176至365位残基组成。活性位点靠近二聚体界面。在其中两种结构中，磷酸吡哆醛辅因子的4'-醛基通过席夫碱连接与活性位点赖氨酸Lys202的ε-氨基共价连接。在第三种结构中，该酶被Tris不可逆地失活。人线粒体BCAT中二聚体的整体折叠与两种细菌酶——大肠杆菌BCAT和D-氨基酸转氨酶（D-AAT）的结构相似。人BCAT和D-AAT假定底物结合口袋内衬的残基完全重排，以允许对具有相反立体化学结构的底物进行催化。就人线粒体支链氨基酸转氨酶而言，第一个单体中Arg143的胍基与第二个单体中Tyr70的侧链羟基之间的氢键相互作用在底物结合口袋的形成中很重要。
