Mechanistic Enzymology of Phosphoryl Transfer Enzymes

Case Western Reserve University, Cleveland OH

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Abstract

DESCRIPTION (provided by applicant): Understanding the mechanisms and transition states of phosphoryl transfer enzymes is important for understanding biological catalysis as well as facilitating the design of novel catalysts and supporting the development of enzyme inhibitors as potential drugs. In solution these reactions can occur by several different mechanisms with characteristic transition states. Transition state protonation, nucleophile and leaving group bonding and overall charge distribution are highly sensitive to the same catalytic modes (electrostatic stabilization, Bronsted acid/base, Lewis acid/base) that are proposed in models of catalysis by phosphoryl transferases. These insights strongly underscore the importance of addressing long standing unanswered questions in the field of biological catalysis: How do the catalytic modes employed by enzymes alter transition state charge distribution? Do enzymes with different active site geometries (isoenzymes) stabilize different transition states? Can differences in transition state structure facilitate the development of competitive inhibitors as potential drugs? Answering these questions will require a mechanistic framework grounded in theory and experiment for solution reactions, and the ability to apply this framework in structure-function studies to determine the transition states and catalytic modes for representative phosphoryl transfer enzymes. A powerful approach to understand enzyme mechanism is by analyzing kinetic isotope effects (KIEs), which measure the differences in ground state and transition state bonding, and integrating this information with molecular and quantum mechanical simulations to evaluate specific mechanistic scenarios and focus experimental efforts. Until now, technical barriers prohibited application of this powerful approach to reaction involving native RNA oligonucleotide substrates of ribozymes and protein phosphoryl transfer enzymes, leaving the questions highlighted above unanswered for an important enzyme class. Now, having established methods for KIE analyses RNA and nucleotide reactions, we using an integrated approach of theory and experiment to gain a comprehensive understanding of the mechanisms of phosphoryl transfer enzymes. The impact of these experiments is amplified by collaboration with Dr. Joseph Piccirilli (U Chicago) and Dr. Darrin York (Rutgers) who provide complementary technical strengths and importantly contribute independent intellectual perspectives. Our combined efforts are directed at providing new insights into how the active site environments of enzymes act to stabilize reaction transition states. The information gained will shed new light on the interplay between active site chemistry and chemical mechanism, which will significantly impact our understanding of biological catalysis and broadly support advances in design of new catalysts and discovery of inhibitors with potential therapeutic application.