Reaction Mechanism of Aspartate Aminotransferase

E.大肠杆菌天冬氨酸氨基转移酶（AspAT）在其活性部位与辅酶磷酸吡哆醛（PLP）结合，催化天冬氨酸氨基转移到PLP的可逆反应，产生磷酸吡哆胺和草酰乙酸。对AspAT及其突变酶进行了X射线晶体学研究，并结合X射线衍射结果和酶动力学分析了该酶的反应机理。AspAT及其与底物类似物的复合物结晶，并使用同步辐射收集衍射数据。在高分辨下测定了AspAT及其配合物的结构。底物与AspAT的结合诱导整个分子的大的构象变化。对活性中心结构的精确分析表明，反应的底物特异性和立体特异性是如何实现的。在底物与AspAT结合时，大的构象变化的驱动力将是底物的构象变化。 ...更多信息 通过释放许多水分子来减少熵。在活性中心中对催化反应起重要作用的氨基酸残基位于PLP的周围。当这些重要的氨基酸残基被其他残基取代时，AspAT的活性显著降低。为了阐明这些重要残基的作用，进行了突变酶的X-射线结构测定。Y 70 F的整体结构与野生型AspAT相似，F70的苯环位置与Y 70相同。用图解法将C_5底物拟合到活性位上，表明该苯环可能识别C_5底物。在Y 70 W中，底物类似物结合活性位点，但不与PLP形成席夫碱。这意味着米氏情结被困住了。R292 V的整体结构与野生型AspAT的结构相似。令人惊讶的是，尽管带电的R292被中性的V292交换，但V292的侧链位于与R292的侧链相似的位置。对Y225 F的X射线衍射分析表明，Y225 F的羟基与PLP之间的氢键对催化反应起着重要作用。少

E. coli aspartate aminotransferase (AspAT) binds the coenzyme pyridoxal phosphate (PLP), in its active site and catalyzes the reversible reaction of transfer of amino group of aspartate to PLP, giving pyridoxamine phosphate and oxaloacetate. X-ray crystallographic study of AspAT and its mutant enzymes was undertaken and the reaction mechanism of this enzyme was analyzed by using the results of X-ray works and enzyme kinetics. AspAT and its complexes with the substrate analogue were crystallized and diffraction data were collected using the synchrotron radiation. The structures of AspAT and the complex were determined at high resolution. The binding of the substrate to AspAT induces the large conformational change of the overall molecule. The precise analysis of the active site structures shows thathow the substrate specifity and the stereospecifity of the reaction are made possible. The driving force of the large conformational change, on binding the substrate to AspAT, will be the inc … More rease of entropy by the release of many water molecules. The amino acid residues which play an important roles on catalytic reaction in the active site are located around PLP. The activity of AspAT decreases markedly when these important amino acid residues are replaced by other residues. In order to elucidate the roles of these important residues, the X-ray structure determination of the mutant enzymes were undertaken. The overall structure of Y70F is similar to that of the wild type AspAT, and the position of the benzene ring of F70 is the same as that of Y70. The fitting Of C_5 substrate into the active site by the graphic method indicates the possibility of the recognition of C_5 substrate by this benzene ring. In Y70W, the substrate analogue binds the active site but does not make schiff base with PLP. This means the Michaelis complex is trapped. The overall structure of R292V is similar to that of the wild type AspAT. Surprisingly, in spite of the exchange of the charged R292 by the neutral V292, the side chain of V292 is located at the similar position to that of R292. X-ray analysis of Y225F shows that the hydrogen bond between OH group of Y225 and PLP are important to the catalytic reaction. Less