Syntheses of (Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and Their inactivation of γ-aminobutyric acid aminotransferase Original Research Article

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid ( and , respectively) were synthesized and investigated as potential mechanism-based inactivators of γ-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core α-trifluoromethyl-α,β-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxycarbonyl)glycinal. Both and inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from or . This inactivating species is released from the active site prior to inactivation, and as a result, and cannot be defined as mechanism-based inactivators. Furthermore, and are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for and , respectively. The enzyme also catalyzes the elimination of the fluoride ion from and . A mechanism to account for these observations is proposed.