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

Abstract

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 ) 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 (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 (about 5...

Direct synthesis of Z-alkenyl halides through catalytic cross-metathesis

“…Alkenyl fluorides are valued because of the importance of organofluorine compounds in medicine 4 , agrochemicals 5 and materials development 6 . A fluoro-substituted olefin can strongly impact the property of a molecule; an example is the Z -fluoroalkene derivative of γ-aminobutyric acid (GABA) transaminase inhibitor 7 , more active than its E isomer 8 yet similarly potent and with a distinct mode of action compared to the parent non-fluorinated alkene (vigabatrin). Fluoro-olefins may be used as substrates in synthesis of fluorine-containing building blocks 9 .…”

Use of fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical advantages

“…In light of earlier success with the simple α-vinyl trigger and Hafnia alvei L-lysine decarboxylase (LDC) [115], we set to examine the α-(2′Z-fluoro)vinyl trigger [116,117] for AADC inactivation in this active site. Figure 13 illustrates the proposed mechanism(s) of AADC inactivation with this new AADC suicide substrate trigger.…”

Mechanism of Inactivation of GABA Aminotransferase by (E)- and (Z)-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid

“…For example, diastereomers 3 and 4 (Figure 1) also differ only as ( E )- and ( Z )-fluoroalkenes, but they have vastly different mechanisms of inactivation of GABA-AT. 19 Because different inactivation mechanisms can occur by minor structural changes, we were interested to determine how 1 and 2 might undergo inactivation of GABA-AT. Furthermore, if they inactivate by a mechanism that disrupts the Arg445-Glu270 salt bridge to provide a new binding pocket, this would confirm the importance of Arg445 in the design of new GABA-AT inactivators.…”