Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage.
Journal Article (Journal Article)
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 (C76 Delta) 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 C76 Delta 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 (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta. 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.
Full Text
Duke Authors
Cited Authors
- Shih, IH; Been, MD
Published Date
- February 13, 2001
Published In
Volume / Issue
- 98 / 4
Start / End Page
- 1489 - 1494
PubMed ID
- 11171978
Pubmed Central ID
- PMC29284
International Standard Serial Number (ISSN)
- 0027-8424
Digital Object Identifier (DOI)
- 10.1073/pnas.98.4.1489
Language
- eng
Conference Location
- United States