Interaction of spin-labeled nicotinamide adenine dinucleotide phosphate with chicken liver fatty acid synthase.
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)
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*
D013113:Spin Labels; D009249:NADP; D064429:Fatty Acid Synthases