Tricarboxylic acid cycle enzymes of the ectomycorrhizal basidiomycete, Suillus bovinus

In crude cell extracts of the ectomycorrhizal fungus, Suillus bovinus, acti vities of citrate synthase. aconitase, isocitrate dehydrogenase. succinate dehydrogenase. fumarase, and malate dehydrogenase have been proved and anal yzed. Citrate synthase exhibited high affinities for both its substrates: o xaloacetate: (K-m = 0.018 mM) and acetyl-CoA (K-m = 0.014 mM). Aconitase sh owed better affinity for isocitrate (K-m = 0.62 mM) than for citrate (K-m = 3.20 mM). Analysis of isocitrate dehydrogenase revealed only small maximum activity (60 nmol x mg protein(-1) x min(-1)), the enzyme being exclusivel y NADP(+)-dependent. Using the artificial electron acceptor dichlorophenol indophenol, activity and substrate affinity of succinate dehydrogenase were rather poor. Fumarase proved Fe2+-independent. Its affinity for malate was found higher (K-m = 1.19 mM) than that for fumarate (K-m = 2.09 mM). High total activity of malate dehydrogenase could be separated by native PAGE in to a slowly running species of (mainly) cytosolic (about 80%) and a faster running species of (mainly) mitochondrial origin. Affinities for oxaloaceta te of the two enzyme species were found identical within limits of signific ance (K-m = 0.24 mM and 0.22 mM). The assumed cytosolic enzyme exhibited af finity for malate (K-m = 5.77 mM) more than one order of magnitude lower th an that for oxaloacetate. FPLC on superose 12 revealed only one activity ba nd at a molecular mass of 100 +/- 15 kDa. Activities of 2-oxoglutarate dehy drogenase and of succinyl-CoA synthetase could not be found. Technical prob lems in their detection, but also existence of an incomplete tricarboxylic acid cycle are considered. Metabolite affinities, maximum activities and pH -dependences of fumarase and of malate dehydrogenase allow the assumption o f a reductive instead of oxidative function of these enzymes in vivo.