Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects.
Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.
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Departments of Biochemistry and Biophysics and Chemistry, Texas A&M University, College Station, Texas 77843-2128, USA.
Fitzpatrick
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D003903:Deuterium / Q000737:chemistry*; D049308:Dioxygenases; D004980:Ethane / Q000031:analogs & derivatives / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005670:Fusarium / Q000201:enzymology; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009595:Nitroparaffins / Q000737:chemistry; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014631:Valerates / Q000737:chemistry / Q000494:pharmacology
D009595:Nitroparaffins; D014631:Valerates; D005182:Flavin-Adenine Dinucleotide; C014705:nitroethane; D003903:Deuterium; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D004980:Ethane