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 K0.5 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.
*School of Biochemistry, Medical School Building, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
Craig T
CT
Armstrong
*School of Biochemistry, Medical School Building, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
J L Ross
JL
Anderson
*School of Biochemistry, Medical School Building, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
Richard M
RM
Denton
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D021122:Protein Subunits; D013045:Species Specificity; D013552:Swine
D007656:Ketoglutaric Acids; D021122:Protein Subunits; D007655:Ketoglutarate Dehydrogenase Complex; D002118:Calcium