The active site of rabbit muscle enolase, a key enzyme on the glycolytic pathway, will be studied. Enolase normally mediates the interconversion of 2-phosphoglyceric acid and phosphoenolpyruvate. Enolase also catalyzes the allylic rearrangement of 2-phosphobutenoic acid to 2-phosphoenol-Alpha-ketobutyrate, but the steric course of the reaction is unknown. This study will attempt to designate the allylic rearrangement as either suprafacial or antarafacial in order to compare the result with the known steric course of the dehydration reaction. Enolase is irreversibly inhibited by both enantiomers of glycidol phosphate but the course of the inactivation has not been determined. This work will attempt to identify the site of alkylation. The overall project will involve a) the resolution of 2-phospho-3-butenoic acid; b) incubation of deuterated 2-phospho-3-butenoic acid in HTO and subsequent analysis of a chiral methyl group; c) the synthesis of each enantiomer of glycidol phosphate labeled with both C-13 and 0-18; and d) C-13 NMR analysis of the resultant inhibition product.
