The thrust of this proposal is to elaborate the mechanism of action of various enzymes that catalyze differing phosphoryl transfer reactions. The experimental approach projects an interplay between complimentary nonenzymic and enzymic investigations with the former serving to define features of possible transition states and the latter designed to elaborate the steps and associated energetics of the reaction sequence, the existence and structure of enzyme bound intermediates, and the nature as well as influence on key steps of enzyme-substrate interactions. The acquired insights should broaden our general perspective of enzymic catalysis. The nonenzymic studies focus on the reactions of thiophosphate monoesters; the enzymic investigations encompass the following activities: the polymerization of nucleoside diphosphates catalyzed by primer independent polynucleotide phosphorylase; the 3'-elongation of a DNA template-primer as well as degradation of the duplex via 3'-5' hydrolysis or pyrophosphorolysis promoted by the multiple activities of DNA polymerase I; and the site specific hydrolytic cleavage accomplished by the endonuclease, EcoRI. Since crystallographic studies now have revealed structural features of the latter two enzymes, experiments have been emphasized that correlate catalytic function with structure. Specific approaches to be employed include pre-steady state and isotope-partitioning kinetic methods, variations in both substrate and protein structures, and distance measurements within substrate enzyme complexes. It is hoped that the results of the proposed studies are also generally applicable to understanding nonchemical phenomenona such as processivity, translocation, and the mode of sequence searching employed by these enzymes.
Showing the most recent 10 out of 36 publications
