Localization of lipoyl-bearing domains in the alpha-ketoglutarate dehydrogenase multienzyme complex.
The alpha-ketoglutarate dehydrogenase complex from Escherichia coli consists of a core component, dihydrolipoyl transsuccinylase (E2), to which are noncovalently bound 12 polypeptide chains each of alpha-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase. E2 exists as a cube-shaped complex comprising 24 identical chains and may be resolved from the other two enzyme components. Limited digestion of E2 with trypsin quantitatively removes domains containing the lipoic acid cofactor while leaving the quaternary structure of the complex intact. Averages of native and trypsin-modified E2 were computed from images of single molecules obtained from electron micrographs of negatively stained specimens. The two averages were very similar and were in general agreement with a model determined previously by X-ray crystallography. However, detailed analysis of the difference image, obtained by subtracting the average of the trypsin-treated E2 from the native E2, showed extra stain-excluding regions along the edges of the native molecule which we interpret as representing the lipoyl-bearing domains. Micrographs of mixtures of native and modified E2 were also analyzed in order to rule out staining or electron-optical artifacts as accounting for the results. On the basis of these results along with other available structural information, we propose that one function of the lipoyl domains is to permit interactions between distantly separated lipoyl moieties in the E2 complex; this proposal also agrees with recent results of modeling studies of biochemical data [Hackert, M.L., Oliver, R.M., & Reed, L.J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2226-2230].
D000217:Acyltransferases / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D007655:Ketoglutarate Dehydrogenase Complex / Q000032:analysis*; D007658:Ketone Oxidoreductases / Q000032:analysis*; D008854:Microscopy, Electron; D008958:Models, Molecular; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase; D014357:Trypsin