title: Interaction of spin-labeled nicotinamide adenine dinucleotide phosphate with chicken liver fatty acid synthase.
abstract: The spatial relationships between the four reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding sites on chicken liver fatty acid synthase were explored with electron paramagnetic resonance (EPR) and spin-labeled analogues of NADP+. The analogues were prepared by reaction of NADP+ with 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid, with 1,1'-carbonyldiimidazole as the coupling reagent. Several esterification products were characterized, and the interaction of the N3' ester of NADP+ with the enzyme was examined in detail. Both 1H13, 14N and 2H13, 15N spin-labels were used: the EPR spectrum was simpler, and the sensitivity greater, for the latter. The spin-labeled NADP+ is a competitive inhibitor of NADPH in fatty acid synthesis, and an EPR titration of the enzyme with the modified NADP+ indicates four identical binding sites per enzyme molecule with a dissociation constant of 124 microM in 0.1 M potassium phosphate and 1 mM ethylenediaminetetraacetic acid (pH 7.0) at 25 degrees C. The EPR spectra indicate the bound spin-label is immobilized relative to the unbound probe. No evidence for electron-electron interactions between bound spin-labels was found with the native enzyme, the enzyme dissociated into monomers, or the enzyme with the enoyl reductase sites blocked by labeling the enzyme with pyridoxal 5'-phosphate. Furthermore, the EPR spectrum of bound ligand was the same in all cases. This indicates that the bound spin-labels are at least 15 A apart, that the environment of the spin-label at all sites is similar, and that the environment is not altered by major structural changes in the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D002645:Chickens; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D064429:Fatty Acid Synthases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D013113:Spin Labels / Q000138:chemical synthesis / Q000378:metabolism*
chemical_list: D013113:Spin Labels; D009249:NADP; D064429:Fatty Acid Synthases