Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.

Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger.

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.