This proposal deals with kinetic studies of systems of enzymes and reactants which follow the direct transfer pathway for the transfer of metabolite from its enzyme site of synthesis to its enzyme site of utilization. The extent to which this mechanism is operative will be assessed by steady-state kinetic studies of the effectiveness with which an enzyme-metabolite complex (E1-M) is competent to serve as substrate in an E2 catalyzed reaction versus the aqueous solvated metabolite. Such a survey will be based primarily on the """"""""enzyme-buffer"""""""" technique which we have already described. Wherever the direct transfer of metabolite is the physiologically relevant pathway, we shall further investigate the details of the coupled direct transfer mechanism via transient rapid stopped-flow kinetic techniques involving substantial concentrations of both E1 and E2. These experiments will be performed under conditions such that the concentrations of enzyme sites (E1 and E2) exceed the concentration of common metabolite; conditions relevant to enzyme and metabolite intermediates in a metabolic pathway. The generality of the direct transfer mechanism will be probed among enzyme pairs and among more extended sequences of enzymes. These sequences are within the glycolytic and TCA cycles, and in the interconnections of these pathways with amino acid-, and fatty acid metabolism. We define a new type of """"""""chiral imprint"""""""" experiment which tests for the possibility of extensive, constrained metabolite. The molecular mechanism for the direct transfer of metabolite will be probed by computer graphic analysis of known three-dimensional structures wherever both direct transfer and structural information is available. The physiological significance of direct transfer which profoundly affect current views of the bioenergetics and regulations of metabolic pathways are discussed in this proposal.
