Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with produ ction of nitrite and hydrogen peroxide. The enzyme is irreversibly inactiva ted by incubation with tetranitromethane, a tyrosine-directed reagent, at p H 7.3. The inactivation is time-dependent and shows first-order kinetics fo r two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is ob served when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor comple x is formed before irreversible inactivation occurs. Values of 0.096 +/- 0. 013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate co nstant for inactivation and the dissociation constant for the reversibly fo rmed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-d irected inactivation. The W-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting t hat treatment with tetranitromethane resulted in nitration of the enzyme. C omparison of tryptic maps of nitroalkane oxidase treated with tetranitromet hane in the presence and absence of valerate shows a single peptide differe ntially labeled in the inactivated enzyme. The spectral properties of the m odified peptide are consistent with nitration of a tyrosine residue. The am ino acid sequence of the nitrated peptide is L-L-N-E-V-M-C- (NO2-Y) -P-L-F- D-G-G-N-I- G-L-R. The possible role of this tyrosine in substrate binding i s discussed.