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.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism; D003903:Deuterium; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology; D012118:Resorcinols / Q000378:metabolism; D014316:Tritium
D005420:Flavoproteins; D012118:Resorcinols; D014316:Tritium; D009249:NADP; D003903:Deuterium; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; D015109:4-Hydroxybenzoate-3-Monooxygenase; C021911:orcinol 2-monooxygenase