THE IMPORTANCE OF BEING RIBOSE AT THE CLEAVAGE SITE IN THE TETRAHYMENA RIBOZYME REACTION

The ribozyme derived from the intron of Tetrahymena thermophila pre-rR NA catalyzes a site-specific endonuclease reaction with both RNA and D NA oligonucleotides. The total transition-state stabilization by the r ibozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all- deoxyribose substrate. Here we show that this effect is specific to th e chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding. Substrates with a series of 2'-substitue nts, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), -F(2'-fluoro-2'-deo xyribo), and -H(deoxyribo), follow a linear free energy relationship b etween the rate of the chemical step of the ribozyme-catalyzed reactio n and the pK(a) of the leaving group, with slope beta(leaving group) a lmost-equal-to -0.8. Because proton donation to the 3'-oxygen atom fro m a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that th is ribozyme does not employ general acid catalysis. The 2'-OCH3 (2'-me thoxy-2'-deoxyribo) substituent does not follow this correlation, appa rently due to steric hindrance within the active site. The rate of cle avage of the 2'-substituted substrates by the ribozyme follows the ord er 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., accelerat ion of the reaction by electron-withdrawing groups. The 2'-OH group pr ovides the largest transition-state stabilization. Because of uncertai nty in the relative effect of the 2'-OH and 2'-H substituents on the p K(a) of the neighboring 3'-oxygen leaving group, we do not discount th e possibility of interactions between the 2'-hydroxyl group and the ri bozyme that further enhance reactivity. Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bon d to an incipient oxyanion at the neighboring 3'-position. This oxyani on is forming as the phosphodiester bond is breaking, explaining why t he stabilization is specific to the transition state. Analogous differ ential hydrogen bonding might be widely used by enzymes to achieve sel ective transition-state stabilization.