Tricarboxylic acid cycle enzymes of the ectomycorrhizal basidiomycete, Suillus bovinus.
In crude cell extracts of the ectomycorrhizal fungus, Suillus bovinus, activities of citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase have been proved and analyzed. Citrate synthase exhibited high affinities for both its substrates: oxaloacetate (Km = 0.018 mM) and acetyl-CoA (Km = 0.014 mM). Aconitase showed better affinity for isocitrate (Km = 0.62 mM) than for citrate (Km = 3.20 mM). Analysis of isocitrate dehydrogenase revealed only small maximum activity (60 nmol x mg protein(-1) x min(-1)), the enzyme being exclusively 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 (Km = 1.19 mM) than that for fumarate (Km = 2.09 mM). High total activity of malate dehydrogenase could be separated by native PAGE into a slowly running species of (mainly) cytosolic (about 80%) and a faster running species of (mainly) mitochondrial origin. Affinities for oxaloacetate of the two enzyme species were found identical within limits of significance (Km = 0.24 mM and 0.22 mM). The assumed cytosolic enzyme exhibited affinity for malate (Km = 5.77 mM) more than one order of magnitude lower than that for oxaloacetate. FPLC on superose 12 revealed only one activity band at a molecular mass of 100 +/- 15 kDa. Activities of 2-oxoglutarate dehydrogenase and of succinyl-CoA synthetase could not be found. Technical problems 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 of a reductive instead of oxidative function of these enzymes in vivo.
Grotjohann
N
N
Lehrstuhl für Stoffwechselphysiologie, Fakultät für Biologie, Universität Bielefeld, Germany. Norbert.Grotjohann@Biologie.Uni-Bielefeld.DE
Huang
Y
Y
Kowallik
W
W
D000154:Aconitate Hydratase / Q000378:metabolism; D001487:Basidiomycota / Q000201:enzymology*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D002952:Citric Acid Cycle; D005649:Fumarate Hydratase / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000378:metabolism
D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D013385:Succinate Dehydrogenase; D002950:Citrate (si)-Synthase; D005649:Fumarate Hydratase; D000154:Aconitate Hydratase