INHIBITION OF PIG-HEART CITRATE SYNTHASE BY CARBOXYLIC-ACIDS STRUCTURALLY RELATED TO OXALOACETATE

The kinetics of inhibition of citrate synthase by a number of dicarboxylic and tricarboxylic acids have been examined. Inhibition parameters providing information about equilibrium constants for ligand‐binding to free enzyme and to the binary enzyme · oxaloacetate complex were determined under Michaelis‐Menten conditions. Ligand‐binding to the binary enzyme · acetyl‐CoA complex was studied by a new kinetic approach, based upon an examination of the effect of inhibitors on the second‐degree rate behaviour of the enzyme. Carboxylic acids may bind to the acetyl‐CoA site (as anions), as well as to the oxaloacetate site (as oxaloacetate analogues). Depending on the relative strength of these interactions, inhibition competitive with either or both of the substrates is observed. Kinetically estimated equilibrium constants for the binding of inhibitors to different enzyme species in the catalytic mechanism are reported. The results obtained show that carboxylic acids, in general, bind non‐cooperatively with the substrates. The affinity of citrate synthase for acetyl‐CoA increases about 10‐fold on the binding of D‐malate. The mechanistic significance of this and previously reported heterotropic cooperativity effects in the citrate synthase system is discussed. It is suggested that the binding of oxaloacetate and certain carboxylic acids containing a D‐malate substructure induces a conformational change of the enzyme. This conformational change serves the purpose of creating a binding site for proper accomodation of the acyl moiety of acetyl‐CoA at the catalytic center of the enzyme. Copyright © 1979, Wiley Blackwell. All rights reserved