Kinetic isotope effects in the oxidation of isotopically labeled NAD(P)H by bacterial flavoprotein monooxygenases
- 16 March 1982
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 21 (6) , 1144-1151
- https://doi.org/10.1021/bi00535a006
Abstract
Three bacterial flavoprotein monooxygenases (p-hydroxybenzoate hydroxylase, orcinol hydroxylase [from Pseudomonas putida] and salicylate hydroxylase) were 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 presteady-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 .apprx. 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 and suggests that these bacterial phenolic monooxygenases balance out internal transition states such that no single barrier is fully rate limiting.This publication has 11 references indexed in Scilit:
- pH Variation of isotope effects in enzyme-catalyzed reactions. 1. Isotope- and pH-dependent steps the sameBiochemistry, 1981
- Flavin analogs as mechanistic probes of adrenodoxin reductase-dependent electron transfer to the cholesterol side chain cleavage cytochrome P-450 of the adrenal cortex.Journal of Biological Chemistry, 1980
- Kinetic and mechanistic studies on the reduction of melilotate hydroxylase by reduced pyridine nucleotides.Journal of Biological Chemistry, 1979
- Kinetic studies on the reaction of p-hydroxybenzoate hydroxylase. Agreement of steady state and rapid reaction data.Journal of Biological Chemistry, 1979
- Reactions of pig heart lipoamide dehydrogenase with pyridine nucleotides. Evidence for an effector role for bound oxidized pyridine nucleotide.Journal of Biological Chemistry, 1979
- The stereochemistry of NADH utilization by the flavoenzyme monooxygenase orcinol hydroxylase.Journal of Biological Chemistry, 1979
- Stereospecificity of the intramolecular association of reduced pyridine coenzymesBiochemistry, 1978
- The stereospecificity of bacterial external flavoprotein monooxygenases for nicotinamide adenine dinucleotideArchives of Biochemistry and Biophysics, 1977
- Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studiesBiochemistry, 1977
- Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase.Journal of Biological Chemistry, 1976