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

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrificatio n of primary or secondary nitroalkanes to the corresponding aldehydes or ke tones with production of hydrogen peroxide and nitrite. The enzyme is irrev ersibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactiv ation is time-dependent and shows first-order kinetics for three half-lives . The second-order rate constant for inactivation is 3.4 +/- 0.06 M-1 min-l . The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic map s of enzyme treated with N-[ethyl-1-C-14]maleimide in the absence and prese nce of valerate shows a single radioactive peptide differentially labeled i n the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser deso rption/ionization time-of-flight mass spectrometry. The cysteine residue wa s identified as the site of alkylation by ion trap mass spectrometry.