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 production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex 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 constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.
Gadda
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Departments of Biochemistry and Biophysics and Chemistry, Texas A&M University, College Station, Texas 77843-2128, USA.
Banerjee
A
A
Fitzpatrick
P F
PF
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D049308:Dioxygenases; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005670:Fusarium / Q000201:enzymology; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013774:Tetranitromethane / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D013774:Tetranitromethane