Steady-state kinetic study of fatty acid synthase from chicken liver.

Publisher Website
Google Scholar
Abstract
The steady-state kinetics of chicken liver fatty acid synthase was studied over the pH range 5.9-8.6 in 0.1 M K2PO4/1 mM EDTA at 25.0.degree. C. The steady-state initial velocity, v, which was determined by measuring the rate of consumption of NADPH spectrophotometrically over a wide range of substrate concentrations, followed the rate law v = kcat[EO], in which Ac-CoA is acetyl-CoA, Mal-CoA is malonyl-CoA, the Kj are Michaelis constants, the Kj,i are inhibition constants, kcat is the turnover number, and [EO] is the total enzyme concentration. The product CoA is an inhibitor at high concentrations but activates the enzyme at low concentrations when the concentration of Ac-CoA is high. The rate law can be derived from a simple multistep mechanism; in terms of this mechanism, the Michaelis constants are lower bounds to the substrate dissociation constants, and the turnover number contains the 1st-order rate constants characterizing the reactions required to produce palmitic acid. Plots of kcat, kcat/KN, kcat/KA and kcat/KM versus pH are bell-shaped. Analysis of the results in terms of 2 ionizable groups indicated that in all cases an ionizable group with an apparent pKa of .apprxeq. 6 is of importance. For kcat and kcat/KN, the apparent pKa of the 2nd ionizable group is .apprxeq. 7.8, whereas for kcat/KA and kcat/KM, it is .apprxeq. 7.