Substrate-Recognition Mechanism of Aromatic Amino Acid Aminotransferase

大多数氨基转移酶对中性和酸性氨基酸都有活性。本研究以芳香族氨基酸氨基转移酶（ArAT）为模型酶，探讨了氨基转移酶的双底物识别机制.酸性氨基酸的ω-和α-羧酸基团分别被ArAT的Arg 292和Arg 386识别。Arg 386-Asn 194-PLP的氢键网络被认为是通过底物结合调节PLP电子状态的关键。这反映了α-羧酸基团是所有氨基酸底物共有的，通过底物结合激活酶应该涉及氨基酸底物的共有结构基序.β-羟基化准底物和野生型和[Tyr 70 *Phe]突变体ArAT的反应分析表明，酸性和芳香族氨基酸以几乎相同的构象与酶结合。这也表明Arg 292的侧链必须根据氨基酸底物的性质（酸性或芳香族）而改变构象。对Arg 292突变酶的研究表明，Arg 292的胍基对于底物的芳环的识别是重要的。这表明Arg 292侧链形成了接受芳环的口袋的一部分，或者残基292的侧链需要胍鎓基团进行构象变化以产生口袋。为了在原子水平上进一步分析底物识别机制，需要对ArAT进行X射线晶体学分析。虽然还没有得到大肠杆菌ArAT的晶体，但发现副球菌ArAT结晶良好，足以用于X射线分析。目前正在对酶本身以及与底物类似物的复合物进行结构分析。

Most Aminotransferases are active toward both neutral and acidic amino acids. This research aims to investigate the dual substrate-recognition mechanism of aminotransferases using aromatic amino acid aminotransferase (ArAT) as a model enzyme.1. The omega- and alpha- carboxylate groups of acidic amino acids are recognized by Arg292 and Arg386 of ArAT,respectively. The hydrogen-bonding network of Arg 386-Asn194-PLP was found to be critical for modulating the electronic status of PLP by substrate binding. This reflects that the alpha-carboxylate group is common to all amino acid substrates, and the activation of enzyme through substrate binding should involve the commom structural motif of amino acid substrates.2. Analysis of the reaction of beta-hydroxylated quasisubstrates and the wild-type and [Tyr70*Phe] mutant ArAT showed that the acidic and aromatic amino acids bind to the enzyme in almost identical conformations. This also indicated that the side chain of Arg292 must take altered conformations depending upon the nature of amino acid substrates, acidic or aromatic.3. Studies on the Arg292 mutant enzymes showed that the guanidinium group of Arg292 is important for the recognition of the aromatic ring of substrates. This indicated either that the Arg 292 side chain forms a part of the pocket that accepts the aromatic ring or that the guanidinium group required for the side chain of residue 292 to undergo the conformational change in order to create the pocket.4. In order to analyze further the mechanism of substrate recognition at an atomic level, X-ray crystallographic analysis of ArAT is required. Although the crystal of E.coli ArAT has not been obtained, Paracoccus ArAT was found to be crystallized well enough to be used for X-ray analysis. Structural analyzes of the enzyme itself and the complexes with substrate analogs are now under way.