Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide.
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.
Gadda
G
G
Departments of Biochemistry and Biophysics and Chemistry, Texas A & M University, College Station, Texas 77843-2128, USA.
Banerjee
A
A
Dangott
L J
LJ
Fitzpatrick
P F
PF
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D049308:Dioxygenases; D005033:Ethylmaleimide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology*; D006207:Half-Life; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D020539:Sequence Analysis, Protein; D014357:Trypsin / Q000378:metabolism; D014631:Valerates / Q000494:pharmacology
D005420:Flavoproteins; D010446:Peptide Fragments; D014631:Valerates; D005182:Flavin-Adenine Dinucleotide; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide