Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: .beta.-ketoacyl reductase and enoyl reductase

Publisher Website
Google Scholar
Abstract
The following reactions catalyzed by chicken liver fatty acid synthase were studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25.degree. C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (.beta.-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first 2 reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as 2 consecutive pseudo-1st-order processes: a mechanism consistent with the acetoacetyl-CoA dependence of the pseudo-1st-order rate constant associated with formation of the acetoacetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 .mu.M, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The acetoacetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 .mu.M. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also were carried out. The results obtained indicate the steady-state reductions proceed primarily through intermediates in which the substrate is not covalently bound, whereas the single turnover reduction of covalently bound intermediates is observed in the transient studies. Acetoacetyl-CoA and crotonyl-CoA can serve as primers for steady-state fatty acid synthesis if malonyl-CoA is a cosubstrate. A theoretical and experimental analysis of the products obtained by reduction of the isolated acetoacetyl-enzyme with limited [3H]NADPH shows the ratio of hydroxybutyryl- to butyryl-enzyme is determined by the initial ratio of the concentration of NADPH to that of covalently bound substrate and the ratio of turnover numbers for .beta.-ketoacyl synthase and enoyl reductase. The ratio of turnover numbers obtained from the product analyses is in good agreement with that obtained from the stopped-flow kinetics. The theoretical treatment can be utilized for any sequence of 2 reactions involving a common substrate.

This publication has 12 references indexed in Scilit: