The pyruvatedehydrogenase complex PDC functions at an important branch point forenergy metabolism in regard to the utilization and storage of cellularfuels--carbohydrate and fat. Mammalian PDC is a giant enzyme complexcomposed of six components with ninedistinct subunits. Five subunitsform an organized structure that allows them to carry out the coordinatedseries of reactions of the complex. Four of the subunits two in akinase and two in a phosphatase function in the regulation of PDC byinterconverting the pyruvate dehydrogenase E1 component between anactive dephospho form and inactive phospho form. Sixtytransacetylase E2 subunits assemble to form a polyhedral innerstructure with the bulk of E2's mass in an extended, highly segmentedouter structure consisting of 3 domains connected by relatively largeand highly mobile linker regions. Two of these are lipoyl domains. Thefocus of this proposal is to understand the highly consequential rolesof lipoyl domains in E1 and kinase function. The lipoyl domains serveas substrates of E1 in the rate limiting second step of PDC catalysisthat generates a thioacetyl and reducing equivalents. The principalinvestigator will characterize how the lipoyl domains interact with E1and will evaluate the likely prospect that phosphorylation blocks E1catalysis by altering this lipoyl domain-E1 interaction. The kinase hasbeen shown to be a member of a new eukaryotic family of kinases relatedto the prokaryotic histidine kinases. It binds tightly to the innerlipoyl domains of E2 by an unusual association involving the lipoylprosthetic group. The proposed work focuses on the molecular mechanismby which the flexibly connected lipoyl domains of E2 operate both tofacilitate enhanced kinase activity and to mediate effector modulationof kinase activity. Studies are designed to explain enhanced functionby dissecting the mechanisms whereby tightly bound kinase rapidly movesbetween lipoyl domains and otherwise has its activity enhanced whenbound just to an isolated lipoyl domain. The principal investigator willalso investigate how a specialized signal translation mechanismfunctions to establish feedback control of mammalian PDC through alipoyl prosthetic group-mediated process that stimulates kinaseactivity. To discriminate the multiple roles played by lipoyl domainsin E1 and kinase function, the principal investigator will employnative, mutated, and prosthetic group modified lipoyl domains as free, carrier-linked, and oligomeric E2 structures. Understanding how PDC isregulated constitutes essential knowledge for diagnosing human diseases. Aberrant PDC operation is associated with diabetes and obesity. Inbornerrors in components cause severe defects. Primary biliary cirrhosisis an autoimmune disease with antibodies to E2 and other PDC subunits.
research seeks to elucidate the short-term regulation of the mammalian PDC
Acetylation, Acyltransferase, Chemical Kinetics, Circular Dichroism, Cofactor, Conformation, Enzyme Activity, Enzyme Complex, Enzyme Mechanism, Enzyme Structure, Enzyme Substrate, Hydropathy, Nuclear Magnetic Resonance Spectroscopy, Phosphorylation, Protein Kinase, Pyruvate Dehydrogenase, Site Directed Mutagenesis, Thermodynamics
