The goals of this research are (1) to understand the roles of allosteric interactions in the regulation of metabolite transfer between sequential enzymes in a metabolic pathway, (2) to understand the relationship between enzyme conformation change, substrate specificity, and catalysis, and (3) to explore the significance of direct metabolite transfer in systems which contain more than one type of catalytic site. Tryptophan synthase systems from E. coli and S. typhimurium (bienzyme complexes) and yeast and blue-green algae (multienzymes) will be studied utilizing rapid mixing kinetic techniques in combination with absorbance and fluorescence spectrophotometry, and high resolution nuclear magnetic resonance. %%% The efficiency of cellular metabolism requires the existence of multienzyme complexes which catalyze a sequence of chemical changes on metabolites. The efficiency derived from not having to recapture intermediates from the exterior milieu is suspected to be the primary reason for such highly organized processing. The proposed research explores an ideal model system for such linked enzyme-catalyzed processing of substrates. In the case of tryptophan synthase, substrate is passed through a "tunnel" between the enzyme active sites. The studies planned will examine how the two active sites interact with each other in order to optimize catalysis and transfer of metabolites between them. A great deal will potentially be learned through this work about the way this complex protein machinery works and why it is designed as it is.
