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Substrate-Recognition Mechanism of Aromatic Amino Acid Aminotransferase

芳香氨基酸转氨酶的底物识别机制

基本信息

批准号：
06680628
负责人：
HAYASHI Hideyuki
金额：
$ 1.47万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06680628/
关键词：
aromatic amino acid aminotransferase substrate specificity pyridoxal 5'-phosphate substrate analog reaction intermediate hydrogen bond guanidinium group X-ray crystallography 大腸菌 Paracoccus denitrificans 基質認識機構酵素反応速度論 X線結晶解析芳香環

项目摘要

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.

大多数转氨酶对中性和酸性氨基酸都有活性。本研究以芳香氨基酸氨基转移酶（ArAT）为模型酶，探讨转氨酶的双底物识别机制。酸性氨基酸的-和-羧酸基团分别被ArAT的Arg292和Arg386识别。Arg 386-Asn194-PLP的氢键网络被发现是通过底物结合调节PLP电子状态的关键。这反映了-羧酸基是所有氨基酸底物所共有的，酶通过底物结合激活应该涉及氨基酸底物的共同结构基序。对β -羟基化准底物与野生型和[Tyr70*Phe]突变体ArAT的反应分析表明，酸性氨基酸和芳香氨基酸以几乎相同的构象结合在酶上。这也表明Arg292的侧链必须根据氨基酸底物的性质（酸性或芳香性）改变构象。对Arg292突变体酶的研究表明，Arg292的胍基对于识别底物的芳香环很重要。这表明Arg 292侧链形成了接受芳香环的口袋的一部分，或者是残基292侧链为了形成口袋而经历构象变化所必需的胍基。为了在原子水平上进一步分析底物识别的机理，需要对ArAT进行x射线晶体学分析。虽然大肠杆菌ArAT晶体尚未获得，但发现ArAT副球菌结晶良好，足以用于x射线分析。对酶本身及其与底物类似物的配合物的结构分析正在进行中。