The pyridine nucleotides NAD and NADP are the most abundant coenzymes in eukaryotic cells, In addition to their well-studied roles in redox reactions, they participate in a variety of non-redox systems, including DNA repair, cell differentiation, Ca2+ mobilization, and phagocytosis. In our study of the NADP-dependent enzyme isocitrate dehydrogenase (IDH) we have synthesized photoreleasable NAD and NADP compounds, with the nicotinamide nitrogen modified with a carboxynitrobenzyl (CNB) caging group. We would like to characterize these compounds to determine their usefulness in biological systems (since the compounds are identical except for a phosphate group, we will perform flash kinetic analysis on only the caged NADP compound). We would like to measure the absorption spectrum of the expected aci-nitro intermediate, and then use this to determine the quantum efficiency and rate constant for decay of the intermediate. We would also like to photolyze the compound in the presence of IDH and monitor the rate of formation of NADPH simultaneously with decay of the aci-nitro species.
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