The flavoprotein d-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of (R)-nicotine, the oxidation of a carbon-nitrogen bond in the pyrrolidine ring of (R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase. There is no solvent isotope effect on the kcat/Kamine value with either (R)-6-hydroxynicotine or the slower substrate (R)-6-hydroxynornicotine. The effect of pH on the rapid-reaction kinetic parameters establishes that only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding. The K348M substitution has only a small effect on the kinetic parameters; the E350A and E350Q substitutions decrease the kcat/Kamine value by ∼20- and ∼220-fold, respectively, and the E352Q substitution decreases this parameter ∼3800-fold. The kcat/Kamine-pH profile is bell-shaped. The pKa values in that profile are altered by replacement of (R)-6-hydroxynicotine with (R)-6-hydroxynornicotine as the substrate and by the substitutions for Glu350 and Glu352, although the profiles remain bell-shaped. The results are consistent with a network of hydrogen-bonded residues in the active site being involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin.
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