Text

theses

Thiamin diphosphate (ThDP)-dependent enzymes [pyruvate dehydrogenase complex (PDHc), 2-oxoglutarate dehydrogenase complex (2-OGDHc) and 1-deoxy-D-xylulose 5-phosphate synthase (DXPS)] form a diverse class of proteins, which play critical roles in catalyzing a vast variety of metabolic reactions, especially the formation of carbon-carbon bonds. Many of their functions rely on complexes rather than on individual components. Understanding how proteins behave in isolation as well as how they recognize their binding partners is therefore critical for understanding the functions and regulation of proteins. In recent decades, our understanding of protein-protein or protein-ligand interactions have moved beyond rigid binding to conformational changes upon binding, multistep ordered assembly, and structural fluctuations occurring within fully assembled complexes. In this thesis for most experiments two methods were utilized: (a) External fluorophores introduced via site-specific covalent labeling to measure dissociation constants (Kd) to establish the strength of interaction; and (b) Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to study the interactions within PDHc, 2-OGDHc and DXPS. First, we designed a fluorescence titration experiment to measure the dissociation constants between different components within PDHc, to quantitatively study the role that each domain plays in interaction with its regulatory enzyme PDKs. We also conducted a series of HDX-MS experiments to investigate the interaction pattern between the first component of PDHc E1p and PDKs in Chapter 3. In Chapter 4, we mainly focus on the binary complex interactions within 2-OGDHc. By using an external site-specifically introduced fluorophore tag, we measured the dissociation constants between different components and with FRET experiments, we also could identify principal interaction loci. In Chapter 5, we used the HDX-MS method for a study on the dynamics of DXPS. Three of all the peptic peptides derived from DXPS displayed the very unique EX1-type hydrogen/deuterium kinetics, which enabled us to understand how some of the DXPS domains respond to different components and identified two likely slowly interconverting conformation in the active center of the enzyme, a closed and an open one, suggesting mechanistic information about this bisubstrate ThDP enzyme. In the Appendix, we report some preliminary data related to the phosphorylation loop in the PDHc E1p component. It shows that peptides located on phosphorylation loop also display EX1-kinetics, which might be a critical property for the catalysis and regulation of thiamin-dependent enzymes. All studies in this thesis are aimed at a better understanding of mechanisms in ThDP catalysis, and we hope that these fundamental research subjects could guide the development of future structure-based inhibitors and perhaps cure some diseases.

ETD_8704

Ph.D.

Includes bibliographical references

by Luying Yang

doi:10.7282/T3GF0XX6

ETD doctoral

The author owns the copyright to this work.