Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage.
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.
Shih
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Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Been
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D001483:Base Sequence; D002021:Buffers; D003596:Cytosine / Q000378:metabolism*; D004791:Enzyme Inhibitors; D003698:Hepatitis Delta Virus / Q000201:enzymology; D007093:Imidazoles; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011522:Protons; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012997:Solvents
D002021:Buffers; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011522:Protons; D016337:RNA, Catalytic; D012997:Solvents; C029899:imidazole; D003596:Cytosine