Analysis of solvent nucleophile isotope effects: evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis.

Heavy atom isotope effects are a valuable tool for probing chemical and enzymatic reaction mechanisms; yet, they are not widely applied to examine mechanisms of nucleophilic activation. We developed approaches for analyzing solvent (18)O nucleophile isotope effects ((18)k(nuc)) that allow, for the first time, their application to hydrolysis reactions of nucleotides and nucleic acids. Here, we report (18)k(nuc) for phosphodiester hydrolysis catalyzed by Mg(2+) and by the Mg(2+)-dependent RNase P ribozyme and deamination by the Zn(2+)-dependent protein enzyme adenosine deaminase (ADA). Because ADA incorporates a single solvent molecule into the product inosine, this reaction can be used to monitor solvent (18)O/(16)O ratios in complex reaction mixtures. This approach, combined with new methods for analysis of isotope ratios of nucleotide phosphates by whole molecule mass spectrometry, permitted determination of (18)k(nuc) for hydrolysis of thymidine 5'-p-nitrophenyl phosphate and RNA cleavage by the RNase P ribozyme. For ADA, an inverse (18)k(nuc) of 0.986 +/- 0.001 is observed, reflecting coordination of the nucleophile by an active site Zn(2+) ion and a stepwise mechanism. In contrast, the observed (18)k(nuc) for phosphodiester reactions were normal: 1.027 +/- 0.013 and 1.030 +/- 0.012 for the Mg(2+)- and ribozyme-catalyzed reactions, respectively. Such normal effects indicate that nucleophilic attack occurs in the rate-limiting step for these reactions, consistent with concerted mechanisms. However, these magnitudes are significantly less than the (18)k(nuc) observed for nucleophilic attack by hydroxide (1.068 +/- 0.007), indicating a "stiffer" bonding environment for the nucleophile in the transition state. Kinetic analysis of the Mg(2+)-catalyzed reaction indicates that a Mg(2+)-hydroxide complex is the catalytic species; thus, the lower (18)k(nuc), in large part, reflects direct metal ion coordination of the nucleophilic oxygen. A similar value for the RNase P ribozyme catalyzed reaction provides support for nucleophilic activation by metal ion catalysis.