This is a competitive renewal request for a proposal that was initiated in 1988. The major goal is to gain a detailed mechanistic understanding of enzymes that catalyze reactions of two distinct classes: specifically enzymes that catalyze nucleophilic substitutions at phosphorous, a reaction that permeates all of biochemistry and molecular biology, as well as enzymes that catalyse enolization reactions. The objective is to elucidate the general principals of the enzyme chemistry and to develop spectroscopic approaches to enzyme structure and mechanism. The principal targets for study to date have been Staphylococcal nuclease, topoisomerase I and the MutT pyrophosphohydrolase. These enzymes accelerate rates of nucleophilic substitutions at phosphorous by factors of 10(9) to 10(16). Also under investigation now are the mechanisms of the enolization reactions catalyzed by ketosteroid isomerase and 4-oxalocrotonate tautomerase (4-OT), which have catalytic powers of 10(10) and 10(8), respectively. High resolution multidimensional NMR, paramagnetic metal probes, nuclear relaxation rates, and Overhauser effects are used to determine the solution structures and dynamics of the enzymes, the roles of essential metal ions, and the conformations, exchange rates and amino acid environments of enzyme-bound substrates and transition state analogs. This information is used to """"""""dock"""""""" the substrate into the NMR (or X-ray) determined structure of the enzyme. Comparisons of pH-rate profiles of enzymes with pKA values of specific residues, directly determined by 1H, 13C, and 15N NMR titrations, are used to identify general acid and base catalysts. The effects of single and double mutations of active site residues on substrate and metal ion affinities and on catalysts provide independent tests of structure and mechanism, as well as quantitative understanding of the chemical origins of the large rate accelerations. In the next review period, it is proposed to focus major efforts on elucidating the mechanisms of topoisomerase I, MutT pyophosphohydrolase, ketosteroid isomerase and 4-OT. Further experimentation is also proposed to test the now highly developed proposed mechanism for Staphylococcal nuclease.
Showing the most recent 10 out of 85 publications
