Studies on Active Site of Transaminase by Site-Directed Mutagenesis

我们通过对位点诱变产生的突变酶的动力学分析，研究了大肠杆菌天冬氨酸转氨酶的一些活性位点残基的功能作用。天冬氨酸转氨酶是许多转氨酶中研究最广泛的代表；x射线分析已经完成。我们的研究结果如下：1。Lys258在催化过程中是必不可少的，它作为一个催化碱从氨基酸底物中提取a -质子，这是催化的先决条件。222位的负电荷和Tyr225的羟基与辅酶未质子化的羟基之间的氢键可能有助于降低辅酶的电子密度，以促进A -质子的去除。Arg292和Arg386对二羧基底物的识别至关重要。将Arg292取代为不带电的残基，大大提高了中性氨基酸的转氨效率。对粘合有什么影响吗？Trpl4O的内环不仅在催化过程中调节辅酶环的旋转运动，还可能参与了二羧酸底物羧基侧链的结合。tyr70的酚基对于稳定与所有底物的过渡态是必不可少的。苯环在70位的存在是识别谷氨酸-2-氧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.