| The energy of ATP hydrolysis is coupled to some very important biological processes. Some ATPases mediate this energy coupling without a direct interaction between ATP and components of the driven process. Elucidation of the mechanisms of this coupling, termed conformational coupling, is one of the most important, yet daunting problems in enzymology and bioenergetics. In this thesis bacterial NAPRTase is presented as a potential model for the mechanism of conformational energy coupling.;The gene coding for Salmonella typhimurium NAPRTase was cloned from a Salmonella typhimurium DNA library. A bacterial system for NAPRTase overproduction was constructed and the enzyme was purified to homogeneity by a new rapid procedure.;Bacterial NAPRTase was found to be a soluble monomeric enzyme with a molecular weight of 45,000. The cloned enzyme catalyzes a 1:1 ATP hydrolysis and NAMN synthesis. ATP hydrolysis shifts the product-to-substrate ratio for NAMN synthesis by a factor of 1000, showing that the two reactions are energetically coupled. A phosphorylated enzyme intermediate has been isolated and characterized as a link between ATP hydrolysis and NAMN synthesis.;The mechanism for conformational energy coupling between ATP hydrolysis and NAMN synthesis reactions catalyzed by bacterial NAPRTase is proposed on the basis of steady state initial velocity measurements and is compared to the mechanisms of energy coupling proposed for Ca... |
