The structures of the pyruvate and Alpha-ketoglutarate dehydrogenase complexes (PDC, KGDC) from bacterial and mammalian sources will be studied by a combination of electron microscopic and biochemical approaches. Electron microscopy will be done on unstained, frozen-hydrated specimens as well as on specimens prepared by conventional negative staining techniques. Wherever possible, computerized image processing methods, including three-dimensional reconstruction, will be applied in order to extract the maximum information from the micrographs and to eliminate subjective interpretations of the images. The locations and the dimensions of the lipoic acid bearing domains in the complexes will be determined. These domains are located on the transacylase (E2) component of the complexes which may be resolved from the other components, E1 and E3, as a multisubunit complex. By quantitatively comparing micrographs of the native E2 with micrographs of E2 from which the lipoyl domains have been removed by proteolysis, the precise locations of the lipoyl domains will be determined. The reach of the lipoic acid moieties will be determined by labeling them with probes sufficiently large so as to be visible in electron micrographs. Intramolecular crosslinking of the lipoic acid moieties in conjunction with electron microscopy and image processing studies is expected to reveal the modes by which the lipoyl domains communicate with one another. The binding locations on the E2 core of the E1 and E3 will be determined by studying subcomplexes consisting of the E2 core and one or a few bound E1 or E3 subunits; the mammalian PDC also contains a few molecules each of a regulatory kinase and phosphatase specific for the E1 component, but bound to the E2 core. It is expected that the proposed structural studies will aid in understanding how KGDC and PDC assemble and function efficiently despite having non-equimolar stoichiometries of the component enzymes. For example, how are only three tightly bound kinase molecules capable of phosphorylating all sixty of the E1 subunits present in the PDC of bovine heart?
