Measurement of distance between the active serine of the thioesterase domain and the pantetheine thiol of fatty acid synthase by fluorescence resonance energy transfer.
Fatty acid synthase from the uropygial gland was inactivated by treatment with pyrenebutyl methanephosphonofluoridate by specific modification of the "active serine" at the thioesterase domain. Treatment of fatty acid synthase with 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin resulted in the loss of the condensation activity and overall synthase activity. Acetyl-CoA and malenyl-CoA protected the enzyme from inactivation by this reagent suggesting that the pantetheine thiol was modified. In support of this conclusion was the finding that modification of the primer-binding thiol with iodoacetamide prior to the modification with the coumarin derivative resulted in no change in the binding of the coumarin to the enzyme. Furthermore, the presumptive active site peptide isolated after proteolysis released its attached coumarin upon treatment with alkali under beta-elimination reaction conditions. Graphical analysis of the binding data suggested that binding of one coumarin derivative/subunit of the synthase would result in complete loss of the synthase activity. When the synthase was modified with the coumarin and pyrene derivatives, fluorescence resonance energy transfer occurred from the pyrene at the thioesterase site to the coumarin attached to the pantetheine thiol. Dissociation of the enzyme to monomers did not decrease the efficiency of transfer, but limited trypsin treatment, which released the thioesterase domain, abolished the fluorescence resonance energy transfer. These results suggested that the energy transfer occurred between intrasubunit sites. The distance between the pyrene at the thioesterase active site and the coumarin attached to pantetheine thiol on the same subunit of fatty acid synthase was estimated from the efficiency of energy transfer to be 37 A.
D002851:Chromatography, High Pressure Liquid; D003374:Coumarins; D004735:Energy Transfer; D064429:Fatty Acid Synthases; D008433:Mathematics; D009943:Organophosphorus Compounds; D010204:Pantetheine; D012694:Serine; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D009943:Organophosphorus Compounds; D012694:Serine; D010204:Pantetheine; C017692:4-(1-pyrenyl)butyl methylphosphonofluoridate; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D064429:Fatty Acid Synthases