Isolation, purification and characterisation of 2-oxoglutarate reductase from Fusobacterium nucleatum.
2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.
Gharbia
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SE
Department of Oral Microbiology, London Hospital Medical College, Whitechapel, U.K.
Shah
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HN
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D004795:Enzyme Stability; D005673:Fusobacterium / Q000201:enzymology*; D013379:Substrate Specificity
D000429:Alcohol Oxidoreductases; C023858:2-hydroxyglutarate dehydrogenase