The mammalian branched-chain dehydrogenase complex (abbreviated branched- chain complex) is a mitochondrial multienzyme system that catalyzes the oxidative decarboxylation of alpha-keto acids derived from the branched-chain amino acids leucine, isoleucine and valine. The enzyme complex organized about a transacylase (E2b) core, consisting of 24 lipoyl-bearing polypeptides, to which a decarboxylase (Elb), a dehydrogenase (E3), a specific kinase and a specific phosphatase are attached through non-covalent interactions. The long-range goal of this project is to understand at the molecular level the structure and function of the mammalian branched-chain complex. The information obtained will have wide implications on macro-molecular organization and function.
Specific aims are: 1) To determine the crystal structure of the E2b inner core. X-ray crystallography will be carried out with highly purified recombinant E2b proteins. The crystal structure of the inner-core domain when solved will be compared with that of E. coli chloramphenicol acetyltransferase type III. This approach will test the hypothesis that the three-dimensional structures of acyl-CoA transferases are conserved during evolution. 2) To define the active site and subunit-binding domains of E2b. Site-directed mutagenesis and serial deletion experiments will be utilized to map the active site and subunit-binding region of E2b. These experiments are aimed to establish His 391 as the active site for the transacylation reaction. Methods will be developed to establish the putative Elb/E3 binding domain. Flexible hinge regions in recombinant E2b will also be studied by 1H-NMR spectroscopy. 3) To investigate the mammalian lipoylation system. We will use a lipoylation assay to locate the subcellular compartment in which the apo-E2b is lipoylated, and to purify and characterize the mammalian lipoylating enzyme. These experiments will test the hypothesis that lipoylation occurs inside mitochondria. 4) To study structure and function of the Elb-component. Recombinant Elb-alpha and Elb-beta subunits will be utilized to study their assembly and their ability to bind and reductively acylate lipoyl-E2b. The putative TPP-binding site on Elb-alpha will be established by site-directed mutagenesis and equilibrium dialysis. These experiments will define the catalytic and structural roles of Elb-alpha and beta-subunits in the branched-chain complex.
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