Hayashi H, Inoue K, Mizuguchi H, Kagamiyama H
Department of Biochemistry, Osaka Medical College, Japan.
Biochemistry. 1996 May 28;35(21):6754-61. doi: 10.1021/bi960390v.
Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromatic amino acids. Because both reactions are supposed to occur in a single reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intriguing question. This was probed by spectroscopic analysis of the complexes of beta-hydroxylated substrates and the wild-type and [Tyr70-->Phe] mutant enzymes. Both L-erythro-3-hydroxyaspartate and L-erythro-3-phenylserine reacted with the wild-type ArAT to give an absorption maximum at around 500 nm, reflecting the formation of the quinonoid intermediate. When the hydroxy group of Tyr70 of ArAT was deleted by replacement of the residue with phenylalanine, the 500-nm absorption greatly decreased in either of the ArAT-beta-hydroxy amino acid complexes, showing the presence of specific interactions, which stabilize the 500-nm absorbing quinonoid intermediates, between the phenolic hydroxy group of Tyr70 and the beta-hydroxy groups of the two quasisubstrates. From these results, it was concluded that the conformations of the two quasisubstrates are essentially identical in their enzyme-bound forms. This implies that the phenyl group of the substrate phenylalanine occupies the same region as that occupied by the beta-carboxyl group of the substrate aspartate, and the region should be near Arg292, the residue that binds the beta-carboxylate group of substrates. The [Arg292-->Ala] or [Arg292-->Leu] mutation increased the Km values for aromatic amino acids 5-10 fold, and the [Arg292-->Lys] mutation increased these values 10-100-fold, without affecting the kcat values. This shows that the side chain of Arg292 is partially involved in the binding of the aromatic ring of substrates to ArAT.
大肠杆菌芳香族氨基酸转氨酶（ArAT）催化二羧酸氨基酸和芳香族氨基酸的转氨反应。由于这两种反应都被认为发生在单一反应中心，因此ArAT是否为两种不同类型的底物侧链提供了替代结合位点一直是一个有趣的问题。通过对β-羟基化底物与野生型和[Tyr70→Phe]突变酶复合物的光谱分析对这一问题进行了探究。L-赤藓糖型-3-羟基天冬氨酸和L-赤藓糖型-3-苯基丝氨酸都与野生型ArAT反应，在500nm左右产生最大吸收，这反映了醌型中间体的形成。当用苯丙氨酸取代ArAT的Tyr70残基从而删除其羟基时，在任何一种ArAT-β-羟基氨基酸复合物中，500nm处的吸收都大幅下降，这表明Tyr70的酚羟基与两种准底物的β-羟基之间存在特异性相互作用，这种相互作用稳定了500nm吸收的醌型中间体。从这些结果可以得出结论，两种准底物在与酶结合的形式下构象基本相同。这意味着底物苯丙氨酸的苯基占据了与底物天冬氨酸的β-羧基相同的区域，该区域应该靠近结合底物β-羧基的Arg292残基。[Arg292→Ala]或[Arg292→Leu]突变使芳香族氨基酸的Km值增加了5至10倍，而[Arg292→Lys]突变使这些值增加了10至100倍，且不影响kcat值。这表明Arg292的侧链部分参与底物芳香环与ArAT的结合。
