The long term objectives of this project are to elucidate the processes involved in the transmission of regulatory signals in biological systems, as a means toward understanding the molecular mechanisms of metabolic control. The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of signal transduction can be related in some manner to protein conformational changes. For example, the conformational changes induced by the binding of insulin or glucagon to their receptors modulate blood sugar levels, or the large quaternary conformational changes of allosteric enzymes regulate metabolic pathways by altering their catalytic activity. During this project period we will concentrate on three systems involved in signal transmission and allosteric control. The allosteric enzymes aspartate transcarbamoylase (ATCase) and fructose 1,6- bisphosphatase (FBPase) are involved in controlling the rates of the pyrimidine and gluconeogenesis pathways, respectively. Both of these enzymes undergo dramatic conformational changes involving loop motions and movements of entire protein subunits for their function. In addition, we will study the cooperative enzyme, dihydroorotase. This enzyme in pyrimidine nucleotide biosynthesis undergoes loop motions that are coordinated with catalytic activity. A variety of approaches will be used to acquire a molecular-level understanding of how these enzymes function. This project directly addresses fundamental questions of how biological signals are transmitted, in general, and how allosteric regulation controls enzymatic activity, in particular.
The specific aims of this application are divided into fundamental and practical components. We will use state of the art techniques involving time-resolved small-angle X-ray scattering and time-resolved X-ray crystallography to capture the details of the effects that signaling molecules have on these enzymatic systems at the atomic level. We will also use strategically placed fluorescent probes in these enzymes, both to monitor the conformational changes and to relate these conformational changes to their function. We will use X-ray crystallography to define each step in the catalytic and allosteric mechanisms of ATCase, including at the moment of bond formation. We will also use these structural data to design highly potent inhibitors of ATCase and FBPase that may be used for the development of new anti-proliferation, anti-malarial and anti-diabetic agents. Understanding of the relationship between conformational changes and allosteric control in these proteins will also help us elucidate the molecular basis of cellular control mechanisms. ? ? The understanding of the atomic level details of the transmission of regulatory signals, which control most biological processes, is critical for our ability to intervene when these controls break down. This application will concentrate on transmission of regulatory signals in enzymes involved in the pathway that produces the precursors of the nucleic acids, and of one of the pathways involved in maintaining blood sugar levels. A molecular level understanding of how these enzymes exert control over their respective pathway will provide a basis for the rational development of new anti-cancer, anti-diabetic and anti-malarial drugs. ? ? ? ?
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