THE MOLECULAR BASIS OF CELLULAR CONTROL MECHANISMS

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DESCRIPTION (provided by applicant): The long term objectives of this project are to understand the processes involved in signal transmission in biological systems. During this project period we will concentrate on two allosteric enzymes, aspartate transcarbamoylase (ATCase) and fructose 1,6-bisphosphatase (FBPase) that control the rates of the pyrimidine and gluceoneogenesis pathways, respectively. Regulation by allosteric enzymes involves the binding of signaling molecules to specific regulatory sites, and this binding induces conformational changes that alter their activity. ATCase regulates the pyrimidine pathway, which is involved in the biosynthesis of the nucleic acid precursors. Since inhibition of ATCase prevents cell proliferation, this enzyme has become a target for the development of anti-cancer drugs. FBPase is involved in the control of gluceoneogenesis, and when this regulation fails altered blood sugar levels result, making this enzyme a target for the development of anti-diabetic drugs. A molecular level understanding of these control enzymes will provide a basis for the rational development of drugs that can be used to regulate these pathways specifically when required. This project directly addresses fundamental questions of signal transmission in general, and allosteric regulation of enzymatic activity in particular. The specific aims of this proposal are to: (i) use time-resolved X-ray scattering to characterize the allosteric transition of ATCase and the transiently-stable intermediate that exists during the allosteric transition, (ii) investigate the mechanism of signal transmission in ATCase using a pyrene-labeled version of enzyme, hybrid regulatory subunits composed of one wild-type chain and one chain unable to bind effectors, and regulatory chains in which the allosteric and zinc domains are conformationally locked by disulfide bond formation, (iii) differentiate between local and global paths of signal transmission in FBPase using hybrids, fluorescence labels, and mass spectrometry to characterize the distribution of ligated states, and (iv) use x-ray crystallography to determine the transmission of the regulatory signal and the role of protein dynamic motions, such as domain closure, and loop movements for this transfer of the regulatory signal.