Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond Original Research Article

esults: Thio-substitution of the nonbridging pro-SP oxygen in the substrate afflicts chemical turnover but not ground state binding whereas thio-substitution of the nonbridging pro-RP oxygen does not affect the kinetics of RNase T1. Site-directed mutagenesis of the catalytic base Glu58 impairs the enzyme’s ability to discriminate both phosphorothioate diastereomers. Glu58Ala RNase T1 cleaves RP and SP phosphorothioate RNA with similar rates. The dependence of the pro-SP thio-effect on the presence of the Glu58 carboxylate evidences a strong rate-limiting interaction between the nonbridging pro-SP oxygen and the catalytic base Glu58 in the wild type enzyme. Conclusions: Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2′-OH group, the nonbridging pro-SP oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism. Article Outline