The overall objective of this research is to extend our knowledge of structure-function relationships in thiamin diphosphate dependent alpha-keto acid decarboxylases; a large and ubiquitous class of enzymes of fundamental importance to basic metabolism. The project focuses on thiamin diphosphate dependent enzymes thal operate within large multienzyme complexes, and in particular, the E1 component of the pyruvate dehydrogenase multienzyme complex (PDHc). Such multienzyme complexes employ substrate channeling as a means of obtaining high efficiency, and few details are known regarding the key required interactions. The target E1 component from the E. coli PDHc complex is a member of the bacterial alpha (2) E1 family, and is highly homologous in sequence with its counterparts in many pathogenic organisms. The broad, long-term objective is to determine, analyze and understand the structure and function of an intact PDHc complex, while the short term objective is to probe the basic E1 reaction and its interactions with the E2 component.
The specific aims are: (1) To analyze protein-ligand complexes containing the PDHc E1 enzyme along with inhibitors, substrates and/or effectors. This includes the catalytic site directed inhibitors thiaminthiothiazolone diphosphate (ThTTDP), methyl acetylphosphinate, 2-oxo-3-butynoic acid, and fluropyruvate; the substrate/acceptor-substrate analogs pyruvate, lipoic acid and epsilon-N-Lipoyllysine (lipoamide); and the effectors guanosine triphosphate (GTP), acetyI-CoAJCoA and NAD+/NADH. (2) To obtain structure-function information for the E. coli apo-E1 enzyme and for other (non-E1) enzymatic components in the PDHc multienzyme complex, and to determine sites of protein-protein interaction required for complex assembly/function. (3) To identify structural effects resulting from single residue mutations in E. coli PDHc E1 at key catalytic site locations. (4) to extend structure-function analysis of PDHc interactions to include related enzymes from other sources, in particular, the PDHc complex from mycobacterium tuberculosis. The major method to be employed involves x-ray crystallographic studies of isolated proteins, protein-ligand complexes and protein-protein complexes.
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