Text

theses

The focus of this study is on the human pyruvate dehydrogenase complex (PDC) consisting of six proteins including thiamin diphosphate (ThDP)-dependent pyruvate dehydrogenase (E1p, 22-heterotetramer), the dihydrolipoamide transacetylase (E2p, with two lipoyl domains), dihydrolipoamide dehydrogenase (E3), a unique E3-binding protein (E3BP) and two regulatory enzymes, the pyruvate dehydrogenase kinases (PDK1-4) and phosphatases (PDP1-2). The flux of pyruvate through PDC is regulated via reversible phosphorylation (inactivation) at E1 by PDK1-4 and reactivation by PDP1-2. Up-regulation of gene expression of PDK isoforms is involved in several forms of cancer, and PDKs may be further activated by PDC by binding to the E2•E3BP core. Therefore, the PDK: E2•E3BP interaction provides new therapeutic targets. In this thesis functional kinetic studies were carried out to demonstrate significant differences in the activation of PDK isoforms by binding to E2•E3BP core: (i) PDK2 does not need activation by E2•E3BP for its efficient functioning, while PDK4 was the least effective of the four isoforms, and could not be further activated by E2•E3BP. Hence, development of inhibitors to the interaction of PDK2 and PDK4 with PDC core is not promising. (ii) The approach to design inhibitors, which interfere with interaction with PDC is indeed promising for PDK1 and PDK3. PDK3 needs E2•E3BP core for activation, an activation best achieved by synergistic combination of E2-derived catalytic domain and tridomain. Recently the Jordan group successfully identified the interaction loci between PDK1 or PDK2 with the E2•E3BP core. The studies described here interrogate the sites on E2•E3BP identified to interact with PDK1 or PDK2 (Glu35, Val198, Glu153, Glu209), in addition to the lipoyl-lysine sites found on L1 (Lys 46) and L2 (Lys 173). Site-directed mutagenesis was employed on the above amino acids, or ‘hot spots’, and E1 phosphorylation by PDK1, PDK2 or PDK3 in the presence of the constructed L1L2S mutants were tested to identify the resulting effects on overall PDC activity. These measurements will confirm whether the ‘hot spots’ interrogated indeed are important for the interaction of E2•E3BP with the PDKs, and will identify ‘real’ hot spots, against which rational drug design could be undertaken. Additionally, the E1o component of 2-oxoglutarate dehydrogenase complex (2-OGDHc), which belongs to the super-family of the 2 oxo acid dehydrogenase multienzyme complexes, was studied in this thesis for its potential application to chiral synthesis. Specifically, the E. coli and human E1o were used to catalyzed chiral synthesis of α-hydroxy ketones by varying both the 2-oxo acid donor and aldehyde acceptor substrates in a carboligase-type reaction.

ETD_7896

Ph.D.

Includes bibliographical references

Includes vita

by Elena Luisa Guevara

doi:10.7282/T3086881

ETD doctoral

The author owns the copyright to this work.