The long-term goal of this project is to describe the biochemical mechanisms of the lipolytic enzymes responsible for dietary fat digestion in newborns and adults. Dietary fats provide a major source of energy, essential fatty acids, a vehicle for fat soluble vitamins, and components of cellular membranes. Fats eaten in excess adversely affect health increasing the risk of cardiovascular disease and the Incidence of some cancers. The digestion of dietary fats depends on the actions of multiple enzymes. This proposal focuses on three related proteins secreted by the pancreas, colipase-dependent triglyceride lipase and two highly homologous proteins, termed the pancreatic lipase related proteins. The expression of the related proteins is predominant in the newborn period whereas expression of pancreatic lipase is predominant in older animals suggesting that these lipases have different roles in dietary fat digestion during development. The overall aim of this grant is to delineate the relationship of lipase structure to lipase function. The first specific aim is to characterize the substrate specificities of the related proteins. Both related proteins will be expressed in tissue culture and purified. Their activity against a variety of substrates will be determined. The second specific aim is to identify functionally important regions of pancreatic lipase and the related proteins. Site- specific mutagenesis of specific amino acids or combinations of amino acids will be done and the mutant proteins will be expressed and characterized for activity, lipid binding, colipase interactions, and interfacial activation. The third specific aim is to define the regions of colipase that mediate binding to lipids and interacting with pancreatic lipase. Specific mutations will be introduced Into colipase and the effect on function determined. The final specific aim is to produce enough recombinant protein for x-ray crystallography and NMR. Together the completion of these aims will provide detailed knowledge about the biochemical mechanism of lipase and increase our understanding of dietary fat digestion.
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