Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca²⁺, adenine nucleotides and NADH, with NADH exerting a major influence on the K_0.5 value for Ca²⁺. We investigated two potential Ca²⁺-binding sites on E1, which we term site 1 (D¹¹⁴ADLD) and site 2 (E¹³⁹SDLD). Comparison of sequences from vertebrates with those from Ca²⁺-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca²⁺, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A¹¹⁴AALA) exhibits an almost complete loss of Ca²⁺ activation. Variant ∆site2 (in which the sequence is replaced with A¹³⁹SALA) shows no measurable change in Ca²⁺ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca²⁺-binding site, which is distinct from other previously described Ca²⁺-binding sites.