Enzyme catalysis involves chemical interactions in the ground state and transition state that are difficult to characterize experimentally.  With Darrin York (Rutgers) and Joseph Piccirilli (U Chicago) we are determining the transition states for solution 2'-O-transphosphorylation reactions and for RNase A and HDV ribozyme catalysis using 18O kinetic isotope effects.  The results reveal how active sites can alter transitions states using combinations of catalytic modes unavailable in solution.   To gain insight into molecular recognition we adapted competitive kinetics for high-throughput kinetic analysis (HTS-KIN).  With Eckhard Jankowsky (CWRU) we are examining the entire sequence space of substrate binding for the protein and RNA subunits of the tRNA processing enzyme ribonuclease P which displays broad specificity.  We find pronounced discrimination between sequence variants and a connection between sequence variation and binding similar to highly specific enzymes.  Both approaches provide data readily incorporated into computational models and offer new windows into enzyme function.

Experimental analysis of molecular recognition and catalysis by ribozymes and ribonucleoproteins: Kinetic isotope effects and high throughput sequencing kinetics

245th National Meeting (2013)

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