Das Subha R, Piccirilli Joseph A
Howard Hughes Medical Institute, Department of Biochemistry & Molecular Biology, University of Chicago, 5841 S. Maryland Avenue, MC1028, Chicago, Illinois 60637, USA.
Nat Chem Biol. 2005 Jun;1(1):45-52. doi: 10.1038/nchembio703. Epub 2005 May 3.
Recent crystallographic and functional analyses of RNA enzymes have raised the possibility that the purine and pyrimidine nucleobases may function as general acid-base catalysts. However, this mode of nucleobase-mediated catalysis has been difficult to establish unambiguously. Here, we used a hyperactivated RNA substrate bearing a 5'-phosphorothiolate to investigate the role of a critical cytosine residue in the hepatitis delta virus ribozyme. The hyperactivated substrate specifically suppressed the deleterious effects of cytosine mutations and pH changes, thereby linking the protonation of the nucleobase to leaving-group stabilization. We conclude that the active-site cytosine provides general acid catalysis, mediating proton transfer to the leaving group through a protonated N3-imino nitrogen. These results establish a specific role for a nucleobase in a ribozyme reaction and support the proposal that RNA nucleobases may function in a manner analogous to that of catalytic histidine residues in protein enzymes.
近期对RNA酶的晶体学和功能分析提出了嘌呤和嘧啶核碱基可能作为一般酸碱催化剂发挥作用的可能性。然而，这种核碱基介导的催化模式一直难以明确确立。在此，我们使用了一种带有5'-硫代磷酸酯的超活化RNA底物，来研究关键胞嘧啶残基在丁型肝炎病毒核酶中的作用。该超活化底物特异性地抑制了胞嘧啶突变和pH变化的有害影响，从而将核碱基的质子化与离去基团的稳定联系起来。我们得出结论，活性位点胞嘧啶提供一般酸催化，通过质子化的N3-亚氨基氮介导质子转移至离去基团。这些结果确立了核碱基在核酶反应中的特定作用，并支持了RNA核碱基可能以类似于蛋白质酶中催化组氨酸残基的方式发挥作用的提议。
