title: Role of dihydrolipoyl dehydrogenase (E3) and a novel E3-binding protein in the NADH sensitivity of the pyruvate dehydrogenase complex from anaerobic mitochondria of the parasitic nematode, Ascaris suum.
abstract: The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum. However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle. Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition. As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition. However, although the free A. suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate. More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition. These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A. suum PDC to NADH inhibition during anaerobiosis.
authors.affiliation: Department of Biological Sciences, The University of Toledo, Toledo, OH 43606-3390, USA.
authors.forename: Sally
authors.initials: S
authors.lastname: Harmych
authors.forename: Robin
authors.initials: R
authors.lastname: Arnette
authors.forename: Richard
authors.initials: R
authors.lastname: Komuniecki
mesh_terms: D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D017165:Ascaris suum / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D003001:Cloning, Molecular; D008058:Dihydrolipoamide Dehydrogenase / Q000502:physiology*; D015801:Helminth Proteins / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010455:Peptides / Q000302:isolation & purification / Q000502:physiology*; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013552:Swine
chemical_list: D015801:Helminth Proteins; D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D009243:NAD; D008058:Dihydrolipoamide Dehydrogenase