Consequences of selective removal of each of the lipoyl domains of recombinant dmydrolipoyl transacetylase component (E2) for the function of the kinase and phosphatase components of human pyruvate dehydrogenase complex (PDC)

D. Yang, T. E. Roche

Research output: Contribution to journalArticlepeer-review

Abstract

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 languageEnglish (US)
Pages (from-to)A1510
JournalFASEB Journal
Volume10
Issue number6
StatePublished - Dec 1 1996
Externally publishedYes

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