Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli

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Abstract
The 2-oxoglutarate dehydrogenase multienzyme complex of E. coli was treated with trypsin at pH 7.0 at 0.degree. C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, their apparent MW falling from 50,000 to 36,000 as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase (EC 1.2.4.2) chains was also observed, whereas the lipoamide dehydrogenase (EC 1.6.4.3) chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and EM. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton NMR spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkble intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. The mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

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