Open AccessIsolation, purification and characterisation of 2‐oxoglutarate reductase from Fusobacterium nucleatum
Author(s) -
Gharbia Saheer E.,
Shah Haroun N.
Publication year - 1991
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1991.tb04676.x
Subject(s) - chemistry , fusobacterium nucleatum , biochemistry , enzyme , alpha ketoglutarate , reductase , acetyl coa , citrate synthase , cofactor , stereochemistry , biology , bacteria , genetics , porphyromonas gingivalis
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 × 10 −5 and 0.4 × 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°C for 10 min, while incubation at 60°C abolished all activity.