pH dependencies of the Tetrahymena ribozyme reveal an unconventional origin of an apparent pKa.
The L-21 ScaI ribozyme derived from the Tetrahymena thermophila pre-rRNA group I intron catalyzes a site-specific endonucleolytic cleavage of RNA, DNA, and chimeric RNA/DNA oligonucleotides: CCCUCUA5 + G-->CCCUCU + GA5. The pH-rate dependence was determined for the reaction of the E.G complex with the oligonucleotide substrate d(CCCUC)r(U)d(A5) [(kcat/Km)S conditions]. Although it was shown that the pH dependence is not affected by specific buffers, there is inhibition by specific monovalent cations. The intrinsic pH-rate dependence is log-linear with slope 1 below pH 7, displays an apparent pKa of 7.6, remains nearly level until pH 8.5, and then begins to fall. Two models to explain the apparent pKa were ruled out: (1) the pKa represents loss of a proton from the nucleophilic 3' OH of G, and (2) the pKa arises from a change in rate-limiting step from a pH-dependent to a pH-independent step. In addition, these models, or others involving a single titration, cannot account for the decrease in activity at high pH. A third, unconventional, model is consistent with all of the data. It involves inactivation of the ribozyme by any of several independent titrations of groups with pKa values considerably higher than the apparent pKa of 7.6. The data are consistent with loss of catalytic function upon release of a proton from any one of 19 independent sites with pKa = 9.4 (the unperturbed pKa of N1 of G and N3 of U in solution). Independent experiments investigating the effect of pH on different reaction steps supported this model and suggested the identity of some of the required protons. This mechanism of inactivation is expected to generally affect the behavior of RNAs at pH values removed from the pKa of the titrating bases.
Knitt
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Department of Biochemistry, B400 Beckman Center, Stanford University, California 94305-5307, USA.
Herschlag
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D000818:Animals; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012269:Ribosomal Proteins; D016808:Tetrahymena thermophila / Q000201:enzymology*; D017186:Titrimetry
D007477:Ions; D016337:RNA, Catalytic; D012269:Ribosomal Proteins; C060232:ribosomal protein L21; C047020:endodeoxyribonuclease ScaI; D015252:Deoxyribonucleases, Type II Site-Specific