G. Gadda et al., Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide, J BIOL CHEM, 275(41), 2000, pp. 31891-31895
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.