Structural and functional studies of allosteric regulation of metabolic enzymes

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Nature has evolved many redundant and complementary mechanisms to regulate cellular functions. Among these, allostery provides the most direct, rapid, and efficient means to sense and alter the concentration of small molecules. In an allosteric process, the functional site of a macromolecule is affected by a regulatory molecule binding to another, distally located site. This process is advantageous in controlling macromolecular function because it allows the regulatory molecules, commonly referred to as effectors, to differ widely from the molecules involved in the functional step. The long-term goals of this NSERC supported program are to identify allosterically regulated enzymes and their effectors, elucidate the molecular forces and mechanisms at work, and explore the significance of these mechanisms in metabolic pathways. I am particularly interested in the enzymes involved in two distinct processes, namely, isoprenoid biosynthesis (produces essential fatty metabolites) and monosaccharide phosphorylation (the first step in simple sugar metabolism). The short-term objectives for the next five years are: I) to identify allosteric effectors of farnesyl pyrophosphate synthase and explore the enzyme's conformational transition; II) to characterize the structure of human mevalonate kinase and identify allosteric inhibitors of the enzyme; and III) to elucidate the molecular and cellular mechanisms of ribokinase regulation by monovalent cations. The proposed studies will involve an array of methods. The enzymes of interest will be produced in genetically manipulated bacterial cells and purified by employing chromatographic techniques. Visualization of the enzymes' molecular structures by X-ray crystallography and assessment of their functional activities by in vitro assays will be the primary means of achieving the research objectives. Other biophysical methods that can indirectly characterize molecular interactions, such as isothermal titration calorimetry, will play a role in providing complementary data. Computational molecular simulations and tissue culture work will be carried out for some aspects of the proposed studies. Despite the mounting evidence that allosteric regulation plays a critical role in every essential biological process, it is still an underappreciated research subject. Many of the best-known examples of allosteric regulation have been discovered decades ago (e.g., hemoglobin), with the majority of allosterically regulated enzymes and their natural effectors yet to be identified. Moreover, the molecular mechanisms by which the fundamental physical properties of macromolecules underpin allostery are still poorly understood. The outcomes of this NSERC-funded research program will help fill these gaps and contribute to advancing the fields of systems biology and molecular biophysics. A deeper understanding of allosteric processes will have powerful implications in applied areas, such as drug discovery and protein engineering.