PH DEPENDENCIES OF THE TETRAHYMENA RIBOZYME REVEAL AN UNCONVENTIONAL ORIGIN OF AN APPARENT PK(A)

The L-21 ScaI ribozyme derived from the Tetrahymena thermophila pre-rR NA group I intron catalyzes a site-specific endonucleolytic cleavage o f RNA, DNA, and chimeric RNA/DNA oligonucleotides: CCCUCUA(5) + G --> CCCUCU + GA(5). The pH-rate dependence was determined for the reaction of the E . G complex with the oligonucleotide substrate d(CCCUC)r(U)d (A(5)) [(k(cat)/K-m)(S) conditions]. Although it was shown that the pH dependence is not affected by specific buffers, there is inhibition b y specific monovalent cations. The intrinsic pH-rate dependence is log -linear with slope 1 below pH 7, displays an apparent pK(a) of 7.6, re mains nearly level until pH 8.5, and then begins to fall. Two models t o explain the apparent pK(a) were ruled out: (1) the pK(a) represents loss of a proton from the nucleophilic 3' OH of G, and (2) the pK(a) a rises from a change in rate-limiting step from a pH-dependent to a pH- independent step. In addition, these models, or others involving a sin gle 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 t itrations of groups with pK, values considerably higher than the appar ent pK(a) of 7.6. The data are consistent with loss of catalytic funct ion; upon release of a proton from any one of 19 independent sites wit h pK(a) = 9.4 (the unperturbed pK(a) of N1 of G and N3 of U in solutio n). Independent experiments investigating the effect of pH on differen t reaction steps supported this model and suggested the identity of so me of the required protons. This mechanism of inactivation is expected to generally affect the behavior of RNAs at pH values removed from th e pK(a) of the titrating bases.