The proposed research examines the thermodynamics of substrate binding to human erythrocyte glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the pentose phosphate pathway. The project's health implication is derived from the importance of G6PD. G6PD maintains the reducing environment of the erythrocyte, and in genetically deficient individuals, it is suspected of contributing to hemolysis. The specific aims of the proposal are to measure quantitatively the number of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) molecules bound to G6PD and the magnitude of the association constant for these two molecules. The project will directly measure the equilibrium quantity of radioactively labeled G6P, or NADP, bound to the enzyme. These measurements will be conducted under pH and concentration of potential regulatory molecules (e.g. ATP) conditions known to influence the enzymatic activity of G6PD. The binding data will be analyzed by direct fitting to a series of binding equations of increasing complexity using a non-linear regression algorithm. The algorithm will yield quantitative values for the number and strength of the ligand binding. Comparison between these measurements will indicate the effect of these ligands on G6PD, and analysis of the binding profiles by a linked-functions approach will provide quantitative estimates of the impact of regulatory molecules upon substrate binding.