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

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Abstract
The kinetics 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 were studied in 0.1 M potassium phosphate-1 mM EDTA, pH 7.0, at 25.degree. C by monitoring changes in NADPH fluorescence with a stopped-flow apparatus. Improved fluorescence detection 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 4 equivalent sites on the enzyme (presumably 2 .beta.-ketoacyl reductase sites and 2 enoyl reductase sites). The bimolecular rate constant is 12.7 .times. 106 M-1 s-1, and the dissociation rate constant is 76.7 s-1, which gives an equilibrium Kd of 6.0 .mu.M. The formation of the acetoacetyl-enzyme and its subsequent reduction by NADPH could be analyzed as 2 consecutive pseudo-first-order reactions by mixing enzyme-NADPH with acetyl-CoA and malonyl-CoA under conditions where [acetyl-CoA], [malonyl-CoA] .mchgt. [enzyme] .mchgt. [NADPH]. From the dependence of the rate of reduction of acetoacetyl-enzyme by NADPH on enzyme concentration, an independent estimate of the equilibrium Kd for NADPH binding to the enzyme of 5.9 .mu.M is obtained, and the rate constant for the reduction is 17.5 s-1. The kinetic studies and analysis of the products obtained with [3H]-NADPH suggest that the dehydration and enoyl reductase reaction. The mechanism of formation of the acetoacetyl-enzyme is complex, but the inhibition of the rate of this process by high concentrations of both acetyl-and malonyl-CoA can be explained semiquantitatively in terms of a relatively rapid equilibration of these substrates with the enzyme followed by a rate-determining condensation reaction with a specific rate constant .gtoreq. 30 s-1. If CoA is scavenged from the reaction mixture, the rate of formation of the acetoacetyl-enzyme is greatly decreased; this indicates CoA is important for rapid formation of the acetoacetyl-enzyme. These results are consistent with those from steady-state kinetic studies.

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