Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site.
The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)
Herschlag
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Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215.
Cech
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D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D005091:Exons; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D013379:Substrate Specificity; D013768:Tetrahymena / Q000235:genetics*
D009843:Oligoribonucleotides; D012322:RNA Precursors; D016337:RNA, Catalytic; D006160:Guanosine Triphosphate