Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage
| Autor: | I-hung Shih, Michael D. Been |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Stereochemistry
Molecular Sequence Data Buffers Cleavage (embryo) Cytosine chemistry.chemical_compound Kinetic isotope effect Side chain RNA Catalytic Enzyme Inhibitors Multidisciplinary Base Sequence biology Chemistry Imidazoles Ribozyme RNA Biological Sciences Rate-determining step Kinetics Mutagenesis Solvents biology.protein Nucleic Acid Conformation RNA Cleavage Hepatitis Delta Virus Protons |
| Zdroj: | Proceedings of the National Academy of Sciences. 98:1489-1494 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.98.4.1489 |
| Popis: | Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D 2 O and H 2 O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k cat / K M conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|