Kinetic isotope effects in the oxidation of isotopically labeled NAD(P)H by bacterial flavoprotein monooxygenases.

Three bacterial flavoprotein monooxygenases, p-hydroxybenzoate hydroxylase, orcinol hydroxylase, and salicylate hydroxylase, have been examined for steady-state kinetic isotope effects with (4R)-[4-2H]NAD(P)H and (4R)-[4-3H]NAD(P)H. The observed isotope selections are for deuterium, DV = 1.8-3.5 and D(V/K) = 1.7-5.1, and for tritium, T(V/K) = 5-19. For both orcinol hydroxylase and p-hydroxybenzoate hydroxylase, reduction of enzyme-bound FAD by (4R)-[4-2H]NAD(P)H in pre-steady-state assays reveals intrinsic deuterium isotope effects of 10 +/- 2 on this redox step. These values are at the upper end of substrate deuterium effects seen in enzymatic reactions. Suppression of approximately 83% of the intrinsic isotope effects in the overall reaction rate (e.g., kH/kD = 10 down to DV = 2.5) corroborates earlier kinetic data on p-hydroxybenzoate hydroxylase [Husain, M., & Massey. V. (1979) J. Biol. Chem. 254, 6657] and suggests that these bacterial phenolic monooxygenases balance out internal transition states such that no single barrier is fully rate limiting.