(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain 7-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 ± 79 fluoride ions; transamination occurs 737 ± 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840–1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.
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