The mechanism of the flavoprotein D-6-hydroxynicotine oxidase: substrate specificity, pH and solvent isotope effects, and roles of key active-site residues.

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 k(cat)/K-amine 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 k(cat)/K-amine value by similar to 20- and , similar to 220-fold, respectively, and the E352Q substitution decreases this parameter similar to 3800-fold. The k(cat)/K-amine-pH profile is bell-shaped. The pK a 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.