Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or
ketones with production of nitrite and hydrogen peroxide. pH and kinetic i
sotope effects with [1,1-H-2(2)]nitroethane have been used to study the mec
hanism 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 V-D(max)
value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. T
he 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 th
e V-D(max) value increases to a value close to the intrinsic value, consist
ent with cleavage of the carbon-hydrogen bond of the substrate being fully
rate-limiting for catalysis in the presence of imidazole.