Mechanism of RNase T-1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond

Background: The microscopic events of ribonuclease (RNase) catalyzed phosph oryl transfer reactions are still a matter of debate in which the contender s adhere to either the classical concerted acid-base mechanism or a more se quential triester-like mechanism. In the case of RNase A, small thio-effect s of the nonbridging oxygens have been invoked in favor of the classical me chanism. However, the RNase T-1 catalyzed transphosphorylation of phosphoro thioate RNA is highly stereoselective. R-P thio-substituted RNA is depolyme rized 60 000 times faster than S-P thio-substituted RNA by this enzyme, whe reas the uncatalyzed cleavage of both substrates occurs at comparable rates . We combined site-directed mutagenesis in the RNase active site and stereo specific thio-substitution of an RNA substrate to probe the intermolecular interactions of the enzyme with the nonbridging pro-S-P oxygen that bring a bout this stereoselectivity of RNase T-1. Results: Thio-substitution of the nonbridging pro-S-P oxygen in the substra te afflicts chemical turnover but not ground state binding whereas thio-sub stitution of the nonbridging pro-R-P oxygen does not affect the kinetics of RNase T-1. Site-directed mutagenesis of the catalytic base Glu58 impairs t he enzyme's ability to discriminate both phosphorothioate diastereomers. Gl u58Ala RNase T-1 cleaves R-P and S-P phosphorothioate RNA with similar rate s. The dependence of the pro-S-P thio-effect on the presence of the Glu58 c arboxylate evidences a strong rate-limiting interaction between the nonbrid ging pro-S-P oxygen and the catalytic base Glu58 in the wild type enzyme. Conclusions: Based on these results, we put forward a new triester-like mec hanism for the RNase T-1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S-P oxygen and o ne of the carboxylate oxygens of Glu58. This interaction allows nucleophili c attack on an activated phosphate to occur simultaneously with general bas e catalysis, ensuring concerted phosphoryl transfer via a triester-like mec hanism.