Many types of tumor cells could theoretically be made sensitive to treatment by L-asparaginase if specific and potent inhibitors of asparagine synthetase were available. However the inhibitors available presently are mainly analogs of glutamine and asparagine that show very little specificity for asparagine synthetase there can be a rational approach to the design of such specific and potent inhibitors there needs to be a complete understanding of the mechanism and chemistry of asparagine synthetase. We have isolated a number of monoclonal antibodies that recognize specific regions of the pancreatic asparagine synthetase that seem responsible for portions of the synthesis reaction, as well as for the glutamine hydrolysis reaction. The epitopes for these, and other antibodies to be isolated, will be mapped by a technique proposed to be generally useful for epitope mapping this relatively large enzymes. The chemical mechanism of asparagine synthesis will be examined by a set of kinetic experiments that will probe partial reactions, as well as the overall reaction mechanism. The genes for the asparagine synthetase from human, bovine pancreas, and E. coli will be cloned and expressed so that site-directed mutagenesis and mechanism based inhibitors can probe the site proposed to be responsible for the glutamine hydrolysis reaction catalyzed by asparagine synthetase. Finally, the sites predicted as being essential in the reactions catalyzed by asparagine synthetase from the epitope mapping and chemical modification studies will be probed by a combination of site-directed mutagenesis and chemical studies that the results can be used to test predictions made by molecular modeling. The result of the proposed program will be detailed chemical picture of asparagine synthetase and the reactions it catalyzes.
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