An aromatization mechanism of inactivation of γ-aminobutyric acid aminotransferase for the antibiotic L-cycloserine

Gregory T. Olson, Mengmeng Fu, Sharon Lau, Kenneth L. Rinehart, Richard B. Silverman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

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 1.1 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 examine-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 structure that is one of the automeric forms of the Schiff base between PMP and oxidized cycloserine (21).

Original languageEnglish (US)
Pages (from-to)2256-2267
Number of pages12
JournalJournal of the American Chemical Society
Volume120
Issue number10
DOIs
StatePublished - Mar 18 1998

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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