Subunit interactions in the mammalian alpha-ketoglutarate dehydrogenase complex. Evidence for direct association of the alpha-ketoglutarate dehydrogenase and dihydrolipoamide dehydrogenase components.
Selective tryptic proteolysis of the mammalian alpha-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its alpha-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and also a fully active E1' fragment. Similarities between the N-terminal region of E1 and the dihydrolipoamide acetyltransferase (E2) and E3-binding components (E3BP) of the pyruvate dehydrogenase complex are highlighted by the specific cross-reactivities of subunit-specific antisera. Analysis of the pattern of release of E1 and E1' polypeptides from the OGDC during tryptic inactivation suggests that both polypeptide chains of individual E1 homodimers must be cleaved to permit the dissociation of the E1 and E3 components. A new protocol has been devised that promotes E1 dissociation from the oligomeric dihydrolipoamide succinyltransferase (E2) core in an active state. Significant levels of overall OGDC reconstitution could also be achieved by re-mixing the constituent enzymes in stoichiometric amounts. Moreover, a high affinity interaction has been demonstrated between the homodimeric E1 and E3 components, which form a stable subcomplex comprising single copies of these two enzymes.
McCartney
R G
RG
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Rice
J E
JE
Sanderson
S J
SJ
Bunik
V
V
Lindsay
H
H
Lindsay
J G
JG
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D008322:Mammals; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation
D046911:Macromolecular Substances; D015636:Magnesium Chloride; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase