The long term objective of this project is to study the stereochemistry and mechanism of biological reactions and interactions involving biophosphates. In the previous two periods 31p NMR Methods have been developed to study the stereochemistry of phosphoryl transfer reactions. Phospholipids labeled with 17O, 18O, or sulfur at the phosphate group have been synthesized and applied to various biochemical problems. This proposal will continue the stereochemical study of phospholipid-related enzymes, and extend the work to biophysical properties of chiral thiophospholipids and their interaction with some enzymes.
Specific aim 1 involves elucidation of the steric courses of the reactions catalyzed by four enzymes: (i) phospholipase C; (ii) choline kinase; (iii) phosphatidylserine synthase; and (iv) phosphatidyl glycerol phosphate synthase.
Specific aim 2 involves synthesis and configuration analysis of chiral thiophosphatidylcholine and chiral thiophosphatidylethanolamine with varying acyl groups, as well as chiral dipalmitoylthiophosphatidylserine and chiral dipalmitoylthiophosphatidylglycerol.
Specific aim 3 involves biophysical properties of chiral thiophospholipids in the liquid crystalline phase, by differential scanning calorimeter, Fourier-transform infrared spectroscopy, 31p NMR, 14N NMR, and 2H NMR.
Specific aim 4 involves stereospecificity of six membrane-related enzymes toward diastereomers of thiophospholipids.
Specific aim 5 is to initiate study on """"""""coenzymes chiral at phosphorus,"""""""" including (i) synthesis of NADHAlphaS and stereospecificity of two dehydrogenases; (ii) synthesis of FADAlphaS and stereospecificity of two oxidases. The information to be obtained may enhance our understanding in the stereochemical mechanism of enzyme catalysis, the interaction between the phosphate group of phospholipids and some membrane-related enzymes.
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