Abstract
Experimental studies on the cleavage of various RNA molecules--poly(U), 3',5''-UpU, 2',5''-UpU, and 3',5''-ApA--catalyzed by imidazole buffers show that there is a sequential bifunctional mechanism. One catalyst species converts the substrate to an intermediate phosphorane, and the second catalyst converts the phosphorane to cleavage products. Detailed steady-state kinetics are presented to determine all mechanisms that are consistent with the findings. Choice among these possibilities can be made considering other experimental evidence about the catalysis of an isomerization reaction that accompanies cleavage. It is concluded that all acceptable mechanisms involve action of the second catalyst on a phosphorane monoanion; it can be formed directly from the substrate in the first step or by proton equilibrations involving either the substrate or the phosphorane intermediate. The relationship of these conclusions to the likely mechanism of action of the enzyme ribonuclease is briefly discussed.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Breslow R., Huang D. L., Anslyn E. On the mechanism of action of ribonucleases: dinucleotide cleavage catalyzed by imidazole and Zn2+. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1746–1750. doi: 10.1073/pnas.86.6.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Breslow R., Xu R. Recognition and catalysis in nucleic acid chemistry. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1201–1207. doi: 10.1073/pnas.90.4.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cleland W. W. Partition analysis and the concept of net rate constants as tools in enzyme kinetics. Biochemistry. 1975 Jul 15;14(14):3220–3224. doi: 10.1021/bi00685a029. [DOI] [PubMed] [Google Scholar]