The broad goal of this project is to understand the specific chemical strategies used by enzymes to catalyze phosphoryl transfer reactions, which is one of the most common and essential reaction in biology. The current focus of our research is on enzymes that are prototypes for defining fundamental features of enzymatic catalysis. Because phosphoryl transfer is essential in biology, enzymes of this class are potential therapeutic targets for human diseases. The results from the current project period will increase understanding of the fundamental principles underlying catalysis setting the stage for studies that may lead to new therapeutics. While the study of phosphoryl transfer is decades old, there remain long standing, fundamental questions about the mechanisms of these enzymes. Crystal structures for many of these enzymes have been solved;yet, significant discrepancies between structures and inconsistencies with functional experiments remain. This lack of understanding is a key barrier to discovering the defining features of biological catalysis by this enzyme class and realizing the potential for design of mechanism-based inhibitors. To overcome this barrier, we developed new methods for analyzing enzyme transition state interactions by measuring kinetic isotope effects. In this method the effect on reaction rate of substituting and atom undergoing reaction with a heavier stable isotope is measured. The magnitude of these effects provides detailed information about the transition state structure. These data can be used to interrogate the interactions between diverse biological catalysts and the transition state for phosphoryl transfer reactions. Our current efforts focus on comparing ribozyme and protein phosphoryl transfer enzymes, aiming to understand their underlying unique and idiosyncratic catalytic strategies. The ability to compare catalysis at the level of transition state charge distribution, combined with the ability to manipulate active site interactions in defined ways provides a powerful means to distinguish enzymatic features involved in specific catalytic mechanisms. Although our main focus is on heavy atom isotope effects, the overall approach is multidisciplinary. Information from high resolution structures, steady state and pre-steady state kinetics with wild type and mutant enzymes, and computational simulations will be integrated to obtain information about the reactions'transition states and their interactions with catalysts.

Public Health Relevance

Cell function depends on enzymes to dramatically increase of the rates of biochemical reactions and they are the target of numerous diagnostics, drugs and clinical therapy. The research will for the first time test directly how enzymes that are important for human health and pathogenesis increase rates of reactions involving the formation and breakage of RNA and DNA chains.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM096000-02
Application #
8329007
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
2011-09-15
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2012
Total Cost
$259,050
Indirect Cost
$94,050
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Chen, Haoyuan; Giese, Timothy J; Huang, Ming et al. (2014) Mechanistic insights into RNA transphosphorylation from kinetic isotope effects and linear free energy relationships of model reactions. Chemistry 20:14336-43
Kellerman, Daniel L; York, Darrin M; Piccirilli, Joseph A et al. (2014) Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2'-O-transphosphorylation. Curr Opin Chem Biol 21:96-102
Lin, Hsuan-Chun; Yandek, Lindsay E; Gjermeni, Ino et al. (2014) Determination of relative rate constants for in vitro RNA processing reactions by internal competition. Anal Biochem 467:54-61
Gu, Hong; Zhang, Shuming; Wong, Kin-Yiu et al. (2013) Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2'-O-transphosphorylation. Proc Natl Acad Sci U S A 110:13002-7
Guenther, Ulf-Peter; Yandek, Lindsay E; Niland, Courtney N et al. (2013) Hidden specificity in an apparently nonspecific RNA-binding protein. Nature 502:385-8
Radak, Brian K; Harris, Michael E; York, Darrin M (2013) Molecular simulations of RNA 2'-O-transesterification reaction models in solution. J Phys Chem B 117:94-103
Wong, Kin-Yiu; Gu, Hong; Zhang, Shuming et al. (2012) Characterization of the reaction path and transition states for RNA transphosphorylation models from theory and experiment. Angew Chem Int Ed Engl 51:647-51