Yennawar Neela H, Conway Myra E, Yennawar Hemant P, Farber Gregory K, Hutson Susan M
Department of Biochemistry and Molecular Biology, Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.
Biochemistry. 2002 Oct 1;41(39):11592-601. doi: 10.1021/bi020221c.
The three-dimensional structures of the isoleucine ketimine and the pyridoxamine phosphate forms of human mitochondrial branched chain aminotransferase (hBCATm) have been determined crystallographically at 1.9 A resolution. The hBCATm-catalyzed transamination can be described in molecular terms together with the earlier solved pyridoxal phosphate forms of the enzyme. The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis. A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place. Short-chain aliphatic amino acid side chains are unable to interact through van der Waals contacts with any of the surfaces whereas bulky aromatic side chains would result in significant steric hindrance. As shown by modeling, and in agreement with previous biochemical data, glutamate but not aspartate can form hydrogen bond interactions. The carboxylate group of the bound isoleucine is on the same side as the phosphate group of the cofactor. These active site interactions are largely retained in a model of the human cytosolic branched chain aminotransferase (hBCATc), suggesting that residues in the second tier of interactions are likely to determine the specificity of hBCATc for the drug gabapentin. Finally, the structures reveal a unique role for cysteine residues in the mammalian BCAT. Cys315 and Cys318, which immediately follow a beta-turn (residues 311-314) and are located just outside the active site, form an unusual thiol-thiolate hydrogen bond. This beta-turn positions Thr313 for its interaction with the pyridoxal phosphate oxygens and substrate alpha-carboxylate group.
人线粒体支链氨基酸转氨酶（hBCATm）的异亮氨酸酮亚胺和磷酸吡哆胺形式的三维结构已通过晶体学方法在1.9埃分辨率下测定。hBCATm催化的转氨基作用可以从分子层面进行描述，同时结合该酶早期解析出的磷酸吡哆醛形式。活性位点赖氨酸Lys202经历了较大的构象变化，并且辅因子的吡啶环在催化过程中倾斜约18度。该酶对L-支链氨基酸的底物和立体特异性的一个主要决定因素是一组疏水残基，它们形成三个疏水表面并将侧链固定到位。短链脂肪族氨基酸侧链无法通过范德华接触与任何一个表面相互作用，而庞大的芳香族侧链会导致显著的空间位阻。如建模所示，并与先前的生化数据一致，谷氨酸而非天冬氨酸能够形成氢键相互作用。结合的异亮氨酸的羧基与辅因子的磷酸基团在同一侧。这些活性位点相互作用在人胞质支链氨基酸转氨酶（hBCATc）模型中基本得以保留，这表明第二层相互作用中的残基可能决定hBCATc对药物加巴喷丁的特异性。最后，这些结构揭示了半胱氨酸残基在哺乳动物BCAT中的独特作用。紧跟β-转角（残基311 - 314）且位于活性位点外侧的Cys315和Cys318形成了一种不寻常的硫醇 - 硫醇盐氢键。这个β-转角将Thr313定位以使其与磷酸吡哆醛的氧原子和底物α-羧基相互作用。
