Mechanism-based inactivation of γ-aminobutyric acid aminotransferase by 3-amino-4-fluorobutanoic acid

• Published in: Bioorganic & medicinal chemistry 1996-09, Vol.4 (9), p.1521-1535
• Main Authors: Silverman, Richard B., Chamberlain Roscher, Cheryl L.
• Format: Article
• Language: English
• Subjects: 4-Aminobutyrate Transaminase - antagonists & inhibitors; Aminobutyrates - pharmacology; Animals; Brain - enzymology; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzyme Inhibitors - pharmacology; Magnetic Resonance Spectroscopy; Swine
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/0968-0896(96)00145-9

The mechanism of inactivation of the pyridoxal 5′-phosphate (PLP)-dependent enzyme γ-aminobutyric acid (GABA) aminotransferase by 3-amino-4-fluorobutanoic acid ( 2) has been investigated. As in the case of the homologue, 4-amino-5-fluoropentanoic acid ( 1), 2 equiv of radiolabeled inactivator become covalently attached to the enzyme, and no transamination, as determined by the lack of conversion of [1- 14C] α-ketoglutarate into [1- 13C] glutamate during inactivation, was observed. In the case of 1, the conclusion was that inactivation was completely the result of modification of the coenzyme and that there was no metabolic turnover; every enzyme molecule catalysed the conversion of one molecule of inactivator to the activated species, which inactivated the enzyme by an enamine mechanism. With 2, however, 6.7±0.7 equiv of fluoride ions were released during inactivation, and it took 7.6±0.7 inactivator molecules to inactivate each enzyme dimer. Since no transamination was occurring, another metabolic event besides inactivation must result from the PLP form of the enzyme. Inactivation of GABA amino-transferase with [1,2- 14C]- 2 produced [ 14C] acetoacetic acid (about 5.5 equiv) as the metabolite. The 1.93±0.25 equiv of radioactivity covalently bound to the enzyme after inactivation with [1,2- 14C]- 2 and gel filtration were completely released by base treatment. HPLC analysis showed that three radioactive compounds, identified as 2, the product of reaction of PLP with acetone ( 3), and the product of reaction of PLP with acetoacetate ( 4), were detected. The release of 3 and 4 and the prevention of release of radioactivity by treatment with sodium borohydride are consistent with the formation of covalent intermediates that have β-carbonyl-like character, such as 6 and/or 7 (Scheme 2). Inactivation of [ 3H] PLP-reconstituted GABA aminotransferase with 2 followed by gel filtration then base denaturation released all of the radioactivity as a mixture of PLP, 3, and 4. Inactivation with [1,2- 14C]- 2 resulted in the release of 1.37 equiv of 14CO 2, which was shown to be the result of decarboxylation of the acetoacetate/ 4 after release from the enzyme. These results are not consistent with a Michael addition mechanism (Scheme 3), but are consistent with inactivation by an enamine mechanism; release of the enamine five out of seven turnovers accounts for the formation of acetoacetate as the metabolite. To account for the detection of PLP and 2 after denaturation,