Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase.
The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees 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 two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-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 microM. 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 have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)
D000090:Acetoacetates / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002645:Chickens; D003437:Crotonates / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism
D000090:Acetoacetates; D000214:Acyl Coenzyme A; D003437:Crotonates; D006885:Hydroxybutyrates; D009249:NADP; D044943:Fatty Acid Desaturases; D064429:Fatty Acid Synthases