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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 [14C]-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 [3H]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 [14C]- and [3H]-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).

The novel 1,25-(OH)D method exhibited excellent correlation with well validated LC-MS/MS assays from two laboratories. Significantly, its 65min turn-around time is quicker, and sample volume smaller (75μl) than current methods.

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