The E2 component of mammalian PDC has four globular domains-a C-terminal inner-core forming domain (I60), an El-binding domain (B), an inner lipoyl domain (L2), and an N-terminal lipoyl domain (Ll)-connected by large flexible linker regions. cDNA encoding mature human E2 was reconstructed both by deleting the LI coding region, to give a cDNA coding for an L2-B-I structure and by removing the L2 coding region and splicing the regions coding for LI and B-I. The resulting plasmids were pressed in E. coli as soluble (L1-B-I)B and (L1-B-I)A assemblages and these were purified. These L1E2 and L2E2 structures were highly lipoylated and retained high E2 activities and El binding capacities. With excess E1+E3, full-sized mature human E2 (hE2), lacking E3BP, gave 4% of the PDC activity obtained with bovine E2-E3BP, but L1E2 or L2E2 only supported about 1.5% suggesting both lipoyl domains are important for PDC catalysis. The activities of pyruvate dehydrogenase kinase (PDK) and pbosphatase (PDF), which are enhanced several-fold by hE2, were only slightly increased by L1E2 but were substantially enhanced by L2E2, yet to an extent still below those by hE2. Thus, the PDK- and PDF-binding L2 domain plays a major role in supporting enhanced regulatory reactions, but the LI domain must contribute to PDK and POP operating in fully activated states. Acetylation of the lipoates stimulated PDK activity 2.5-fold with hE2, only slightly with L1E2, and 4.3-fold with L2E2, thereby raising PDK activity to that attained with acetvlated-hE2. Whatever role LI has in aidine PDK function, it is bypassed upon acetylation of L2.
|Original language||English (US)|
|State||Published - Dec 1 1996|