Unusual Mechanistic Difference in the Inactivation of γ-Aminobutyric Acid Aminotransferase by (E)- and (Z)-4-Amino-6-fluoro-5-hexenoic Acid (1996)

The mechanisms of inactivation of γ-aminobutyric acid (GABA) aminotransferase by (Z)- (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 [7-3H]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 [3H]-2 or [3H]-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 β-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, and 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. The results for 2 can be rationalized by three different mechanisms. All are initiated by Schiff base formation of the inactivator with the active site PLP followed by γ-proton removal. The major pathway (about 65%) proceeds by isomerization of the fluorovinyl double bond (Scheme 2) and produces a ternary complex between the enzyme, the coenzyme, and the inactivator (13); a small amount of inactivation (about 3%) may result from formation of a weakly stable covalent adduct with an active site nucleophile (14). The other two inactivation pathways proceed by isomerization of the aldimine double bond of the Schiff base with PLP. Scheme 7 (about 30%) results in the formation of a weakly stable adduct that decomposes upon denaturation at neutral pH to 4-amino-6-oxohexanoic acid (31). Scheme 6 (about 5%) could account for the formation of PMP, although this amount may be outside experimental error. The 430 nm absorbance observed in the absorption spectrum may correspond to the conjugated ternary complex 13, which has a structure similar to that of the known product of inactivation of GABA aminotransferase by 35 and 4-amino-5-hexenoic acid (37). The same mechanisms of inactivation by 3 are relevant except that, to avoid the formation of 10, an SN2 mechanism (Scheme 3) is invoked in place of the elimination mechanism (Scheme 2). This difference in mechanism may be the result of hydrogen bonding of the fluorine atoms in different orientations at the active site (Scheme 10). The inactivation mechanisms for 2 and 3, therefore, are different from each other as well as from 4-amino-5-fluoro-5-hexenoic acid (Silverman, R. B.; Bichler, K. A.; Leon, A. J. J. Am. Chem. Soc. 1996, 118, 1241−1252).