Steyaert J
Dienst Ultrastruktuur, Vlaams Interuniversitair instituut Biotechnologie, Vrije Universiteit Brussel, Belgium.
Eur J Biochem. 1997 Jul 1;247(1):1-11. doi: 10.1111/j.1432-1033.1997.t01-1-00001.x.
During the last decade, protein engineering has been used to identify the residues that contribute to the ribonuclease-T1-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2'-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the 2'-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and Phe100 further stabilize the transition state of the reaction. A functionally independent subsite, including Asn36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease T1 guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base.
在过去十年中，蛋白质工程已被用于确定对核糖核酸酶T1催化的酯交换反应有贡献的残基。组氨酸40、谷氨酸58和组氨酸92以高度协同的方式加速了进入的2'-氧下游鸟苷对磷酸原子的亲核取代反应。在质子化的组氨酸40咪唑的辅助下，谷氨酸58从2'-氧中夺取一个质子，而组氨酸92使离去基团质子化。酪氨酸38、精氨酸77和苯丙氨酸100进一步稳定了反应的过渡态。一个功能独立的亚位点，包括天冬酰胺36和天冬酰胺98，通过在离去基团结合时使底物相对于催化侧链排列来促进化学周转。一个涉及残基42-46的不变结构基序通过一系列分子间氢键使核糖核酸酶T1具有鸟嘌呤特异性。酪氨酸42通过平行的面对面堆积相互作用对鸟嘌呤结合有显著贡献。酪氨酸45通常被称为鸟嘌呤结合位点的盖子，对碱基的结合没有贡献。
