The long-term objective of this project is to study the stereochemistry and mechanism of biological reactions and interactions involving biophosphates. In the previous three periods, P-31 NMR and O-17, O-18 or sulphur labeled compounds have been used to elucidate the steric course and stereospecificity of several enzymes. Phospholipids chirally labeled at phosphorus have been synthesized and used to probe the mechanism of interactions between phospholipid molecules and other membrane components, between phospholipid substrate and phospholipases, and between platelet activating factor and its receptor. This proposal will continue and extend the work in three Specific Aims.
In Specific Aim 1, the stereochemistry of six phospholipid-metabolizing enzymes will be investigated: phosphatidylserine synthase from E. coli and yeast, and cytidine diphosphate-diacylglycerol hydrolase from E. coli. These are direct continuation from the third Period. The various substrates have to be synthesized, but the experimental approaches have been well established.
Specific Aim 2 tackles the stereochemistry of physiologically important and mechanistically unique PI-PLC (phosphatidylinositol specific phospholipase C). [O-17, O-18]DPPI (dipalmitoylphosphatidylinositol) and DPPsl the corresponding thiophospho analogue of known configuration will be synthesized and subjected to hydrolysis by PI-PLC. Both products, inositol-1-phosphate(IP) and inositol-1,2-cyclic-phosphate(cIP), will be isolated and their configurations analyzed. Chiral [O-16, O-17, O-18]IP of known configuration will be synthesized by an independent route and used to establish the configurational analysis for IP and cIP. Three distinctive types of PI-PLC, one from B. cereus and two from ram seminal vesicles, will be purified and their mechanisms examined and compared.
In Specific Aim 3, the stereochemical mechanism of PI synthase from yeast will be elucidated by use of techniques developed in specific aims 1 and 2. The information to be obtained in this proposal will be useful in understanding detailed mechanisms of these membrane-related enzymes, and in designing mechanism- based inhibitors for the physiologically important PI-PLC. The stereochemical results on PI-PLC will allow determination of not only whether there is a covalent enzyme-substrate intermediate, but also whether the two products IP and cIP are formed parallelly or sequentially.
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