Regulation of citrate synthase from the citric acid-accumulating fungus, Aspergillus niger.

Citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2cOO leads to acetate-CoA), EC 4.1.3.7) was purified 66-fold from cell-free extracts of a citric acid producing strain of Aspergillus niger. The enzyme is labile at low ionic strength, but can effectively be stabilized by K+, oxaloacetate or glycerol. It has a molecular weight of 80 000 and an optimum pH of 8.5. The enzyme is activated by monovalent cations in dilute buffer solutions, and inhibited by Mg2+ independent of the buffer molarity. Kinetic analysis indicated that the reaction proceeds by an ordered sequential mechanism. The Michaelis constants are: 5 microM for oxaloacetic acid at all concentrations of acetyl-CoA; 10 microM for acetyl-CoA at infinite concentrations of oxaloacetate. Coenzyme A is inhibitory, being competitive with acetyl-CoA (Ki = 0.15 mM) and non-competitive with oxaloacetate. Citrate has no effect. Among various metabolites tested, only ATP can inhibit the enzyme. The inhibition is competitive with acetyl-CoA (Ki = 1.0 mM), and non-competitive with oxaloacetate. Mg2+ partially relieves this inhibition. Other adenine nucleotides are also inhibitory, but to a lesser extent. It is proposed that citrate synthase from Aspergillus niger is only weakly regulated, its activity being mainly controlled by oxaloacetate availability.