Mechanisms of inactivation of gamma-aminobutyric acid aminotransferase by 4-amino-5-fluoro-5-hexenoic acid

An investigation of the mechanisms of inactivation of the pyridoxal 5'-phosphate (PLP)-dependent pig brain gamma-aminobutyric acid (GABA) aminotransferase by 4-amino-5-fluoro-5-hexenoic acid (2), a monofluorinated analogue of the anticonvulsant drug vigabatrin, is described. Inactivation of [H-3]PLP-reconstituted GABA aminotransferase with 2 followed by denaturation released the coenzyme in two forms, one as PLP and the other in a modified form in the ratio 7:3. All enzyme activity was lost upon inactivation by 2, but about 30% of the activity returned upon incubation with PLP, consistent with the formation and release of 30% of the coenzyme in a modified form, as noted above. Inactivation of GABA aminotransferase with [2-H-3]-2 followed by gel filtration resulted in the attachment of 0.7 equiv of tritium to the enzyme, even though complete inactivation occurred. This also is consistent with the above results that about 30% of inactivation is the result of release of a modified coenzyme, leaving 30% of the enzyme as its apoenzyme form.]isolation and mass spectral analysis of the modified coenzyme gave peaks consistent with a modified coenzyme formed from a reaction with the inactivator (27). Denaturation of the enzyme containing 0.7 equiv of radioactivity from the above experiment led to release of 0.2-0.3 equiv of the radioactivity as gamma-acetyl-GABA (20). Treatment of the denatured enzyme with sodium periodate generated 0.2-0.25 equiv of succinic acid, leaving 0.15 equiv of radioactivity still covalently bound to the enzyme. Analysis of amine metabolites shows the formation of 0.5 equiv of 20. Analysis of the nonamine metabolites resulted in the identification of 1 equiv of 4-oxo-5-hexenoic acid (24). After inactivation, 2.6 +/- 0.1 equiv of fluoride ions was detected, consistent with the loss of 1 fluoride ion to produce inactivation, 1 fluoride ion to generate the 4-oxo-5-hexenoic acid, and 0.5 fluoride ion released in the production of gamma-acetyl-GABA. Normal transamination also occurs; 6.3 +/- 0.6 transamination events occurred during inactivation, as measured by the conversion of [C-14]-alpha-ketoglutarate to [C-14]glutamate. These results indicate that there are, at least, three different inactivation mechanisms in effect (Schemes 4-6). All of these mechanisms begin with Schiff base formation between 2 and the active site PLP followed by removal of the gamma-proton and elimination of the fluoride ion. It is from this conjugated allene intermediate (17) that all of the inactivation pathways and metabolites result, except for the normal transamination product. The partition ratio, the amount of inactivator converted to a product per inactivation event, is about 8; 6.5 transaminations, 0.5 conversion to 20, and 1.0 conversion to 24 per 1.0 inactivation event.