Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii
Evidence for the formation of a precatalytic complex with 2-oxoglutarate
Article first published online: 18 AUG 2003
European Journal of Biochemistry
Volume 267, Issue 12, pages 3583–3591, June 2000
How to Cite
Bunik, V., Westphal, A. H. and de Kok, A. (2000), Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. European Journal of Biochemistry, 267: 3583–3591. doi: 10.1046/j.1432-1327.2000.01387.x
- Issue published online: 18 AUG 2003
- Article first published online: 18 AUG 2003
- (Received 17 January 2000, accepted 10 April 2000)
- 2-oxoglutarate dehydrogenase;
- multienzyme complex;
- precatalytic complex;
- Azotobacter vinelandii
The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nm, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mm, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mm substrate inhibition is observed.
From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10−6 to 10−5 m), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10−4 to 10−3 m) the relative contribution of the 2-oxo group in the binding increases.
A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site.
The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.