The goals of this project are to investigate the chemical mechanisms of two bacterial enzymes, LL-diaminopimelate epimerase (E.C.5.1.1.7) and meso-diaminopimelate decarboxylase (E.C.4.1.1.20), and to synthesize """"""""suicide substrate"""""""" inhibitors for them. Such mechanism-based irreversible inactivators may be effective broad spectrum antibiotics since these enzymes are not present in mammals, and the normal metabolic products of these enzymes (DL-diaminopimelate and L-lysine) are utilized in construction of bacterial peptidoglycan cell walls. The research plan includes isolation of the enzymes by established biochemical procedures and chemical synthesis of isotopically-labelled diaminopimelates to serve as substrates in mechanistic studies, especially determination of the stereochemistry of the decarboxylase. Sodium boro[3H]hydride reduction of the enzymes will be used to check for conformational changes occurring upon substrate binding, to confirm the requirement for pyridoxal phosphate in the epimerase, and to aid identification of the amino acid residues in the active sites. A series of diaminopimelates bearing fluorine at the 3 and 5 positions will be chemically synthesized and examined for mechanism-based inactivation of these enzymes. The carbon-14 labeled compounds will also be prepared. All of the fluorinated diaminopimelates will undergo preliminary tests for inhibition of bacterial growth.
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