An Aromatization Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase for the Antibiotic l-Cycloserine

The mechanism of inactivation of pig brain γ-aminobutyric acid (GABA) aminotransferase by the antibiotic l -cycloserine was investigated. l -Cycloserine is a time-dependent inactivator of GABA aminotransferase; no enzyme activity returns upon gel filtration or dialysis. Treatment of GABA aminotransferase with [ 14 C]- l -cycloserine, followed by rapid gel filtration, gives enzyme containing l.l equiv of radioactivity bound. Dialysis or denaturation by acid, base, or urea releases the radioactivity. Inactivation of [ 3 H]pyridoxal 5‘-phosphate (PLP)-reconstituted GABA aminotransferase with l -cycloserine followed by dialysis or denaturation also leads to the release of radioactivity from the enzyme. Both the released [ 14 C]- and [ 3 H]-labeled adducts comigrate by HPLC, suggesting that the inactivation adduct is a condensation product of l -cycloserine with the PLP coenzyme. By HPLC comparison, it was shown that the radiolabeled adduct is not PLP, PMP, PLP oxime, or 4-[3-hydroxy-2-methyl-5-(phosphooxymethyl)-4-pyridinyl]-2-oxo-3-butenoic acid ( 20 ), the expected product of an enamine-type inactivation mechanism. On the basis of the stability of the released adduct to acid and base and its UV−visible spectrum, which has the appearance of a PMP analogue, a simple Schiff base between PLP and cycloserine also was excluded. HPLC of the cycloserine−coenzyme adduct had a retention time very similar to that of the gabaculine−coenzyme adduct. Electrospray ionization tandem mass spectrometry of the isolated cycloserine−coenzyme adduct is consistent with a structure that is one of the tautomeric forms of the Schiff base between PMP and oxidized cycloserine ( 21 ).