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.
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Research Article|
March 28 2014
Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site
Craig T. Armstrong;
Craig T. Armstrong
*School of Biochemistry, Medical School Building, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
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J. L. Ross Anderson;
J. L. Ross Anderson
*School of Biochemistry, Medical School Building, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
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Richard M. Denton
Richard M. Denton
1
*School of Biochemistry, Medical School Building, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
1To whom correspondence should be addressed (e-mail r.denton@bristol.ac.uk).
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Biochem J (2014) 459 (2): 369–381.
Article history
Received:
December 19 2013
Revision Received:
February 04 2014
Accepted:
February 05 2014
Accepted Manuscript online:
February 05 2014
Citation
Craig T. Armstrong, J. L. Ross Anderson, Richard M. Denton; Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site. Biochem J 15 April 2014; 459 (2): 369–381. doi: https://doi.org/10.1042/BJ20131664
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