Studies on Active Site of Transaminase by Site-Directed Mutagenesis

我们通过对定点突变产生的突变酶进行动力学分析，研究了大肠杆菌天冬氨酸氨基转移酶部分活性部位残基的功能作用。天冬氨酸氨基转移酶是许多转氨酶中研究最广泛的代表，已经进行了X射线分析。我们的研究结果如下：1.Lys258是催化所必需的，作为从氨基酸底物上提取a-质子的催化碱基，这是催化的先决条件。位于222位的负电荷以及Tyr225的羟基和辅酶的未质子化羟基之间的氢键可能有助于降低辅酶的电子密度，从而促进a-质子的去除。Arg292和Arg386是识别二元酸底物所必需的。用Arg292取代不带电荷的残基，大大提高了中性氨基酸转氨基的催化效率。对装订有任何影响。Trpl4O的内醚环不仅在催化过程中调节辅酶环的旋转运动，还可能参与二元酸底物的羧基侧链的结合。Tyr7O的苯酚基团对于所有底物的过渡态的稳定是必不可少的。位置70的苯环的存在是识别谷氨酸-2-氧代戊二酸底物对所必需的。

We have examined the functional role of some active site residues of E. coli aspartate aminotransferase by using kinetic analysis on mutant enzymes produced by the site-directed mutagenesis. Aspartate aminotransferase has been the most extensively studied representative of many transaminases ; X-ray analysis has been done. Our findings are as follows :1. Lys258 is essential for catalysis, acting as a catalytic base to withdraw an a -proton from the amino acid substrate, which is a prerequisite for the catalysis.2. A negative charge at position 222, and a hydrogen bond between the hydroxyl group of Tyr225 and the unprotonated hydroxyl group of the coenzyme probably help in lowering the electron density of the coenzyme to facilitate the a- proton removal.3. Arg292 and Arg386 are essential for the recognition of the dicarboxylic substrates.4. Substitution of Arg292 to uncharged residues greatly enhanced the catalytic efficiency of the transamination of neutral amino acid without. any effect on the binding.5. The endole ring of Trpl4O not only regulates the rotational movement of the coenzyme ring during the catalysis, but it may be also involved in the binding of the carboxyl side chain of the dicarboxylic acid substrates.6. The phenol group of Tyr7O is essential for the stabilization of the transition states with all substrates. The presence of the benzen ring at position 70 is necessary to recognize the glutanate-2-oxoglutarate substrate pair.