Poster abstracts

Poster number 73 submitted by Dan Kellerman

Determining mechanisms of enzymatic and non-enzymatic catalysts of RNA 2’-O-transphosphorylation using kinetic isotope effects

Dan Kellerman (Dept. of Biochemistry / RNA Center, Case Western Reserve University), Hong Gu, Shuming Zhang (Dept. of Biochemistry / RNA Center, Case Western Reserve University), Qing Dai (Dept. of Chemistry, University of Chicago), Joseph Piccirilli (Dept. of Chemistry, University of Chicago), Vernon Anderson (National Institute of General Medical Scienes, Bethesda, MD), Mike Harris (Dept. of Biochemistry / RNA Center, Case Western Reserve University)

Abstract:
Transition state theory defines catalysis as the preferential stabilization of a transition state relative to the ground state. Thus, the proficiency of an enzyme is directly related to its ability to recognize and stabilize the transition state. Characterizing the geometry, bonding, and charge distribution of the transition state is therefore critical for understanding catalysis. Despite decades of research on the mechanisms of ribozyme and protein catalysis of RNA strand cleavage by 2’-O-transphosphorylation, there remain significant ambiguities regarding the specific catalytic modes that are used and the roles of active site residues. To help resolve these issues, we are investigating the interactions of enzymatic and non-enzymatic catalysts with the transition state using kinetic isotope effects. The effects on reaction rate of substituting atoms undergoing reaction with heavier, stable isotopes provides information on transition state structure and the interactions with catalysts. We have developed new methods to synthesize RNAs containing site specific isotopic substitutions and for determining the effects of isotopic substitution on reaction rate constants. KIEs for RNA 2’-O-transphosphorylation are providing new insights and the necessary mechanistic clarity for understanding enzyme catalysis. Results to date demonstrate that catalysis by hydroxide occurs by nucleophilic activation by specific base catalysis. KIEs for acid catalysis support modes in which this reaction proceeds by a stepwise mechanism with a phosphorane intermediate, and in which the protonation of a non-bridging phosphoryl oxygen helps to stabilize the intermediate. Results from metal ions catalyzed transphosphorylation reactions indicate that catalysis occurs by leaving group stabilization thorough direct coordination, or by general acid catalysis. Results for RNase A demonstrate a concerted, rather than stepwise mechanism and are consistent with interactions with both the nucleophile and leaving group, but suggest that leaving group bonding is influenced by active site protonation. Together the results demonstrate the range of mechanisms and transition states that are possible for RNA reactions and reveal how catalytic interactions influence transition state bonding.

References:
Harris ME, Dai Q, Gu H, Kellerman DL, Piccirilli JA, Anderson VE. Kinetic isotope effects for RNA cleavage by 2\'-O- transphosphorylation: nucleophilic activation by specific base. J Am Chem Soc. 132(33):11613-21 (2010).

Dai Q, Frederiksen JK, Anderson VE, Harris ME, Piccirilli JA. Efficient synthesis of [2\'-18O]uridine and its incorporation into oligonucleotides: a new tool for mechanistic study of nucleotidyl transfer reactions by isotope effect analysis. J Org Chem. 73(1):309-11 (2008).

Keywords: kinetic isotope effect