The relationships between the catalytic activities of the component enzymes and the overall activity of the α -ketoglutarate dehydrogenase multienzyme complex from Escherichia coli were studied; the spatial relationships between specific sites also were determined with energy-transfer measurements. Titration and inactivation of the α -keto-glutarate decarboxylase component with thiamin thiazolone pyrophosphate indicated that the inactivation of the decarboxylase and the overall activity were aproximately proportional; 12.6 decarboxylase catalytic sites/enzyme complex of molecular weight 2.5ËŸ106 were found. Specific modification of the lipoic acids on the transsuccinylase component with N-[3H]ethylmaleimide revealed 9.12 nmol of maleimide per mg of protein (22.8 lipoic acids/enzyme complex; 1 lipoic acid/polypeptide chain). The loss of overall activity and the extent of modification of lipoic acids were directly proportional, and the labeling reaction was first order. Virtually full activity was found with only three-fourths of the native enzyme FAD (~8 FAD/enzyme complex out of a total of 12). Fluorescence resonance energy transfer measurements between the catalytic site of the α-ketoglutarate decarboxylase labeled with the active site generated probe methyl 4-(1-pyrene)butyryl-phosphonate and FAD or lipoic acid labeled with N-[4-(di-methylamino)-3, 5-dinitrophenyl]maleimide indicated that the FAD and lipoic acids are 33 and 32 Å, respectively, from the decarboxylase catalytic site. The distance between labeled lipoic acids was found to be in the range 24-41 Å, and the distance between N-(3-pyrene)maleimide-labeled lipoic acid and FAD was determined to be 22 Å. In contrast to the results obtained with the pyruvate dehydrogenase multienzyme complex, these distances and the amount of lipoic acid are consistent with a normal catalytic cycle involving the rotation of a single lipoic acid between the catalytic sites. However, apparently both the decarboxylase and the FAD are able to service two or more lipoic acids during catalysis. © 1979, American Chemical Society. All rights reserved.
