The mechanisms of inactivation of gamma-aminobutyric acid (GABA) aminotransferase by (2)- (2)- and (E)-4-amino-6-fluoro-5-hexenoic acid (3) were studied. The kinetic constants of inactivation for 2 and 3 were approximately the same. Inactivation of [7-H-3]PLP-reconstituted GABA aminotransferase by 2 and 3 also gave similar results for the two isomers : 63% (2) and 66%(3) of the radioactivity remained covalently attached to the enzyme; 31% (2) and 29% (3) were released as PLP; 5% (2) and 4% (3) of the radioactivity emerged as PMP. Treatment of GABA aminotransferase with either [H-3]-2 or [H-3]-3 led to the incorporation of 1.0 equiv of tritium into the enzyme after gel filtration. Urea denaturation at pH 7.0, however, released about 0.3 equiv of the tritium from the enzyme, and urea denaturation at pH 2.4 released 0.35 equiv of the tritium. About 85% of the released radioactivity was identified as 4-amino-6-oxohexanoic acid (31) and the remainder as a mixture of 4-oxo-5-hexenoic acid (10) and the product of Michael addition of beta-mercaptoethanol to 10. Neither inactivator produced any amine metabolites during inactivation. The first divergence from similarity between the two isomers was in the isolation of nonamine metabolites. Inactivator 2 generated two nonamine metabolites, whereas 3 produced only one. The additional metabolite with 2 was identified as 10. The metabolite in common may be the normal transamination product or something derived from it. To confirm this possibility, it was shown that both isomers undergo transamination; 2 is transaminated 1.4 +/- 0.3 times, and 3 is transaminated 0.7 +/- 0.3 time. Fluoride ion release also was monitored, and it was found that 2 released 1.4 +/- 0.2 F- and 3 released 0.9 +/- 0.05 F-. The additional F- release for 2 is expected, given that it produces an additional metabolite that requires release of F- for its formation. Absorption spectra of GABA aminotransferase inactivated with 2, 3, aid 4-amino-5-fluoropentanoic acid (35) showed an absorbance at 430 nm that was missing in the spectrum of native enzyme. Taken together, these results indicate that 2 and 3 inactivate GABA aminotransferase by multiple mechanisms, but at least the major inactivation mechanism is different for the two isomers. : 63% (2) and 66%(3) of the radioactivity remained covalently attached to the enzyme; 31% (2) and 29% (3) were released as PLP; 5% (2) and 4% (3) of the radioactivity emerged as PMP. Treatment of GABA aminotransferase with either [H-3]-2 or [H-3]-3 led to the incorporation of 1.0 equiv of tritium into the enzyme after gel filtration. Urea denaturation at pH 7.0, however, released about 0.3 equiv of the tritium from the enzyme, and urea denaturation at pH 2.4 released 0.35 equiv of the tritium. About 85% of the released radioactivity was identified as 4-amino-6-oxohexanoic acid (31) and the remainder as a mixture of 4-oxo-5-hexenoic acid (10) and the product of Michael addition of beta-mercaptoethanol to 10. Neither inactivator produced any amine metabolites during inactivation. The first divergence from similarity between the two isomers was in the isolation of nonamine metabolites. Inactivator 2 generated two nonamine metabolites, whereas 3 produced only one. The additional metabolite with 2 was identified as 10. The metabolite in common may be the normal transamination product or something derived from it. To confirm this possibility, it was shown that both isomers undergo transamination; 2 is transaminated 1.4 +/- 0.3 times, and 3 is transaminated 0.7 +/- 0.3 time. Fluoride ion release also was monitored, and it was found that 2 released 1.4 +/- 0.2 F- and 3 released 0.9 +/- 0.05 F-. The additional F- release for 2 is expected, given that it produces an additional metabolite that requires release of F- for its formation. Absorption spectra of GABA aminotransferase inactivated with 2, 3, aid 4-amino-5-fluoropentanoic acid (35) showed an absorbance at 430 nm that was missing in the spectrum of native enzyme. Taken together, these results indicate that 2 and 3 inactivate GABA aminotransferase by multiple mechanisms, but at least the major inactivation mechanism is different for the two isomers.