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)