Neurospora crassa α-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties

• Published in: Biochimica et biophysica acta Vol. 990; no. 1; pp. 66 - 72
• Main Authors: Bessam, Hassiba, Mareck, Alain M., Foucher, Bernard
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 27.01.1989
• Subjects: Calcium; Calcium - pharmacology; Catalysis; catalytic activity; Cations, Divalent; Coenzyme A; Coenzyme A - metabolism; Component resolution; Electrophoresis, Polyacrylamide Gel; enzyme activity; enzymology; Hydrogen-Ion Concentration; isolation & purification; Ketoglutarate Dehydrogenase Complex; Ketoglutarate Dehydrogenase Complex - isolation & purification; Ketoglutarate Dehydrogenase Complex - metabolism; Ketoglutaric Acids; Ketoglutaric Acids - metabolism; Ketone Oxidoreductases; Ketone Oxidoreductases - metabolism; Kinetic parameter; Magnesium; Magnesium - pharmacology; metabolism; Molecular Weight; N. crassa; NAD; NAD - metabolism; Neurospora; Neurospora - enzymology; Neurospora crassa; Neurospora crassa - enzymology; oxidoreductases; pharmacology; purification; Temperature; Thiamine Pyrophosphate; Thiamine Pyrophosphate - metabolism; α-Ketoglutarate dehydrogenase complex; PEG; N. crassa; E 2; E 1; α-Ketoglutarate dehydrogenase complex; E 3; Component resolution; Kinetic parameter
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/S0304-4165(89)80013-3

A method is proposed for the purification of the Neurospora crassa α-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-poly-acrylamide gel electrophoresis as E 3, E 1 and E 2 with respective M r values of 54000, 53 000 and 49000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca 2+ in particular was investigated. It was shown that Ca 2+ diminishes the K m value of the N. crassa α-ketoglutarate dehydrogenase complex for α-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.