Investigation of reduced nicotinamide adenine dinucleotide phosphate and acyl-binding sites on avian fatty acid synthase.
The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to chicken liver fatty acid synthase has been studied by using both fluorescence titrations and the direct binding method of forced dialysis. Four apparently identical sites are found per enzyme molecule, with an intrinsic dissociation constant of 0.6 microM at pH 7.0, 23 degrees C. The acyl-binding sites on the enzyme have been studied with a fluorescent analogue of acetyl-CoA, beta-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)]alanyl coenzyme A (NBDA-CoA). The enzyme slowly transfers NBDA to acyl-binding sites. Analysis of the kinetics of binding and of the stability and hydroxylamine sensitivity of the acyl-enzyme at pH 7.5 suggests that binding occurs predominantly at two classes of sulfhydryl sites, with two sites of each class per enzyme molecule. Up to one NBDA per enzyme molecule is bound to a nonsulfhydryl site after overnight incubation of enzyme with NBDA-CoA. The acyl linkage at one class of sulfhydryl sites appears to be hydrolyzed by the thioesterase activity of the enzyme. This hydrolysis can be prevented by modifying the enzyme with tosyl fluoride. The binding of NBDA is inhibited by acetyl-CoA, malonyl-CoA, and NADPH. The NBDA-enzyme adduct is inactive, although activity can be partially restored by incubation at 35 degrees C. The binding of NADPH to the enzyme is not significantly altered by the binding of NBDA. Fluorescence resonance energy transfer between enzyme-bound NADPH and enzyme-bound NBDA suggests that the acyl-binding sites are 30-40 A from NADPH-binding sites. This distance can only be defined approximately because of the presence of multiple energy donors and acceptors and the uncertainty in the dipole-dipole orientations of the energy acceptors and donors.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000109:Acetylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004735:Energy Transfer; D064429:Fatty Acid Synthases / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism*
D009249:NADP; C019495:NBD-5-acylcholine; D064429:Fatty Acid Synthases; D009327:4-Chloro-7-nitrobenzofurazan; D000109:Acetylcholine