201. Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide.
- Author
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Gadda G, Banerjee A, Dangott LJ, and Fitzpatrick PF
- Subjects
- Amino Acid Sequence, Binding Sites drug effects, Chromatography, High Pressure Liquid, Cysteine chemistry, Flavin-Adenine Dinucleotide metabolism, Flavoproteins chemistry, Flavoproteins metabolism, Half-Life, Kinetics, Mass Spectrometry, Molecular Sequence Data, Oxygenases antagonists & inhibitors, Peptide Fragments chemistry, Peptide Fragments metabolism, Peptide Mapping, Sequence Analysis, Protein, Trypsin metabolism, Valerates pharmacology, Cysteine metabolism, Dioxygenases, Ethylmaleimide metabolism, Fusarium enzymology, Oxygenases chemistry, Oxygenases metabolism
- Abstract
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 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(-)(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-(14)C]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.
- Published
- 2000
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