Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide

Giovanni Gadda, Ari Banerjee, Lawrence J. Dangott, Paul F. Fitzpatrick

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12 Scopus citations


The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is timedependent and shows first-order kinetics for three halflives. The second-order rate constant for inactivation is 3.4 ± 0.06 M-1 min-1. The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated with N-[ethyl-1-14C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.

Original languageEnglish (US)
Pages (from-to)31891-31895
Number of pages5
JournalJournal of Biological Chemistry
Issue number41
StatePublished - Oct 13 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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