Kittendorf Jeffrey D, Sgraja Tanja, Reuter Klaus, Klebe Gerhard, Garcia George A
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Chuirch Street, Ann Arbor, MI 48109-1065, USA.
J Biol Chem. 2003 Oct 24;278(43):42369-76. doi: 10.1074/jbc.M304323200. Epub 2003 Aug 8.
tRNA-guanine transglycosylase (TGT) catalyzes a post-transcriptional base-exchange reaction involved in the incorporation of the modified base queuine (Q) into the wobble position of certain tRNAs. Catalysis by TGT occurs through a double-displacement mechanism that involves the formation of a covalent enzyme-RNA intermediate (Kittendorf, J. D., Barcomb, L. M., Nonekowski, S. T., and Garcia, G. A. (2001) Biochemistry 40, 14123-14133). The TGT chemical mechanism requires the protonation of the displaced guanine and the deprotonation of the incoming heterocyclic base. Based on its position in the active site, it is likely that aspartate 264 is involved in these proton transfer events. To investigate this possibility, site-directed mutagenesis was employed to convert aspartate 264 to alanine, asparagine, glutamate, glutamine, lysine, and histidine. Biochemical characterization of these TGT mutants revealed that only the conservative glutamate mutant retained catalytic activity, with Km values for both tRNA and guanine 3-fold greater than those for wild-type, whereas the kcat was depressed by an order of magnitude. Furthermore, of these six TGT mutants, only the TGT(D264E) was capable of forming a TGT.RNA covalent intermediate; however, unlike wild-type TGT, only hydroxylamine is capable of cleaving the TGT(D264E).RNA covalent complex. In an effort to better understand the unique biochemical properties of the D264E TGT mutant, we solved the crystal structure of the Zymomonas mobilis TGT with the analogous mutation (D280E). The results of these studies support two roles for aspartate 264 in catalysis by TGT, protonation of the leaving guanine and deprotonation of the incoming preQ1.
转运RNA-鸟嘌呤转糖基酶（TGT）催化一种转录后碱基交换反应，该反应涉及将修饰碱基 queuine（Q）掺入某些转运RNA的摆动位置。TGT的催化作用通过双置换机制发生，该机制涉及形成共价酶-RNA中间体（Kittendorf, J. D., Barcomb, L. M., Nonekowski, S. T., and Garcia, G. A. (2001) Biochemistry 40, 14123 - 14133）。TGT的化学机制需要被置换鸟嘌呤的质子化和进入的杂环碱基的去质子化。基于其在活性位点中的位置，天冬氨酸264可能参与这些质子转移事件。为了研究这种可能性，采用定点诱变将天冬氨酸264分别转换为丙氨酸、天冬酰胺、谷氨酸、谷氨酰胺、赖氨酸和组氨酸。对这些TGT突变体的生化特性分析表明，只有保守的谷氨酸突变体保留了催化活性，其对转运RNA和鸟嘌呤的Km值比野生型大3倍，而催化常数kcat降低了一个数量级。此外，在这六个TGT突变体中，只有TGT(D264E)能够形成TGT.RNA共价中间体；然而，与野生型TGT不同，只有羟胺能够切割TGT(D264E).RNA共价复合物。为了更好地理解D264E TGT突变体的独特生化特性，我们解析了具有类似突变（D280E）的运动发酵单胞菌TGT的晶体结构。这些研究结果支持天冬氨酸264在TGT催化过程中的两个作用，即离去鸟嘌呤的质子化和进入的前Q1的去质子化。
