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

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- 14 C] α-ketoglutarate into [1- 13 C] 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- 14 C]- 2 produced [ 14 C] 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- 14 C]- 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 [ 3 H] 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- 14 C]- 2 resulted in the release of 1.37 equiv of 14 CO 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, it is suggested that a nonproductive formation of the Schiff base of PLP with 2 occurs in the second subunit of the enzyme; this complex is released and hydrolysed to PLP and 2 upon base denaturation. 3-Amino-4-fluorobutanoic acid inactivates γ-aminobutyric acid (GABA) aminotransferase by an enamine mechanism, but five out of every seven turnovers it releases the enamine, producing acetoacetic acid.