Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: acetylation-deacetylation

Abstract

The kinetics of the reaction of acetyl CoA (AcCoA) with fatty acid synthase was studied with a modified quenched-flow technique in 0.1 M potassium phosphate (pH 7.0), 0.5 mM EDTA, and 10% glycerol (wt/vol) at 23.degree. C. The kinetics of the deacetylation of the isolated acetylated enzyme by CoA also was studied. An overall mechanism consistent with the data is .**GRAPHIC**. where E represents the enzyme. The equilibrium dissociation constants, K1 and K3, were estimated to be 85 and 70 .mu.M, respectively, and the rate constants k2 and k-2 are 43 and 103 s-1, respectively. The maximum number of acetyl groups bound to the enzyme in terms of this mechanism is 3.8 (mol/mol). This mechanism also is consistent with the amount of acetylated enzyme formed during titrations of the enzyme and radioactive AcCoA with CoA. The spontaneous hydrolysis of the enzyme at 23.degree. C has a rate constant of 4.7 .times. 10-4 s-1. The acetyl groups on the native enzyme are rapidly removed by hydroxylamine. However, 0.39 of the acetyl groups remains after treatment with hydroxylamine if the enzyme is first denatured in 4 M urea. The acyl binding sites on the native enzyme may be an unstable acetyl oxygen ester and an acetyl thio ester. Destruction of the thioesterase activity of the enzyme through chemical modification of the enzyme does not alter the rate of spontaneous hydrolysis of the acetyl-enzyme nor its reactivity toward hydroxylamine.