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. Here, 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 K0.5 for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (114DADLD) and site 2 (139ESDLD). 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 six-fold decrease in sensitivity for Ca2+, while variant ∆site1 (in which the sequence of site 1 is replaced with 114AAALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with 139ASALA) 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.

Source: BJ Energy - February 5, 2014 Category: Biochemistry Authors: C T. Armstrong, J Ross Anderson, R M. Denton Tags: BJ Metabolism Source Type: research