title: Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: reduced nicotinamide adenine dinucleotide phosphate binding and formation and reduction of acetoacetyl-enzyme.
abstract: The kinetics of reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding to fatty acid synthase from chicken liver and of the reduction of enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase) and the steps leading to formation of the acetoacetyl-enzyme have been studied in 0.1 M potassium phosphate-1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, at 25 degrees C by monitoring changes in NADPH fluorescence with a stopped-flow apparatus. Improved fluorescence detection has permitted the use of NADPH concentrations as low as 20 nM. The kinetics of the binding of NADPH to the enzyme is consistent with a simple bimolecular binding mechanism and four equivalent sites on the enzyme (presumably two beta-ketoacyl reductase sites and two enoyl reductase sites). The bimolecular rate constant is 12.7 X 10(6) M-1 s-1, and the dissociation rate constant is 76.7 s-1, which gives an equilibrium dissociation constant of 6.0 microM. The formation of the acetoacetyl-enzyme and its subsequent reduction by NADPH could be analyzed as two consecutive pseudo-first-order reactions by mixing enzyme-NADPH with acetyl-CoA and malonyl-CoA under conditions where [acetyl-CoA], [malonyl-CoA] much greater than [enzyme] much greater than [NADPH]. From the dependence of the rate of reduction of aceto-acetyl-enzyme by NADPH on enzyme concentration, an independent estimate of the equilibrium dissociation constant for NADPH binding to the enzyme of 5.9 microM is obtained, and the rate constant for the reduction is 17.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D064429:Fatty Acid Synthases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009249:NADP; D010084:Oxidation-Reduction
chemical_list: D009249:NADP; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D064429:Fatty Acid Synthases