Subunit composition and partial reactions of the 2-oxoglutarate dehydrogenase complex of Acetobacter xylinum.

The 2-oxoglutarate dehydrogenase complex from Acetobacter xylinum consists of three components, 2-oxoglutarate dehydrogenase (E1), lipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3). The molecular weight of the complex as studied by sedimentation and diffusion is 2.6 × 106. The E1 component and the E2E3 subcomplex were purified by ultracentrifugation and gel filtration after dissociation of the complex at pH 9.8. The E2E3 subcomplex was further dissociated in 1 M NaCl at neutral pH and the components purified by gel filtration. The active E1 component is a tetramer of Mr 360000. The E3 component is a dimer of Mr 103000. The E2E3 subcomplex showed an S20,w value of 36.6 S indicating an Mr around 1.6 × 106. The Mr of E2 was estimated to be 900000. From these data and the chain weights as derived from sodium dodecylsulphate gel electrophoresis it was concluded that the complex consists of 12 E1, 12 E2 and 12 E3 chains. Incorporation of N-ethyl[2,3-14C]maleimide in the presence of NADH indicated the presence of 12 reactive SH groups per complex located on the E2 component. Titration of the lipoyl moiety with substoichiometric amounts of N-ethylmaleimide in the presence of NADH, as assayed by cleavage with 5,5′-dithio-bis(2-nitrobenzoic acid), indicated that the complex contains 12 lipoyl groups of which only one of the two SH groups is reactive towards N-ethylmaleimide. In agreement with this number, reaction of the complex with 2-oxo[2-14C]glutarate yields incorporation of 12–14 mol succinyl groups/mol complex. With non-saturating concentrations of 2-oxo-glutarate, the presence of the allosteric effector AMP or addition of N-ethylmaleimide was required for maximum incorporation. AMP caused enhancement of the rate of succinylation with cooperative dependence on the AMP concentration, Hill coefficient h= 1.3 at pH 7.4 and h= 2 at pH 8.2. N-Ethylmaleimide prevented desuccinylation. The rate of succinylation was at least six times slower than the rate of FAD reduction as measured from the decrease in fluorescence in the presence of coenzyme A. This indicates that either coenzyme A participates in the succinylation of the lipoyl moiety or that the formation of succinyllipoate is a side reaction of the catalytic pathway, which takes only place in the absence of coenzyme A.