title: The reductive half-reaction of liver microsomal FAD-containing monooxygenase.
abstract: The addition of substrates to microsomal FAD-containing-monooxygenase (EC 1.14.13.8) is an ordered process in which FAD reduction by NADPH is necessarily the first step. This reduction by NADPH is markedly biphasic, and when analyzed for two phases, rate constants of 2.46 s-1 and 0.88 s-1 are obtained at 4 degrees C, pH 7.2. The Kd (8 microM) for the binding of NADPH to the flavoprotein prior to reduction demonstrates a tight binding. Reduction by NADH also occurs and is similar in all respects to that observed with NADPH with the exception that the Kd is larger (0.167 mM) and easily measured. Oxygenatable substrates pre-equilibrated with the enzyme do not change the reduction in any way. NADP+ equilibrated with the enzyme prior to reduction removes the faster phase of the reaction leaving only the slower 0.88 s-1 phase. However, NADP+ rapidly mixed with the enzyme at the time of reduction does not affect the biphasic nature of the reduction, indicating that the binding of NADP+ to the enzyme results in a comparatively slow change of the form of the enzyme responsible for the fast phase into that which exhibits the slower rate. A primary deuterium isotope effect of approximately 6-fold has been observed on both phases of the reduction using (4R)-[4-2H]NADPH. This is strong evidence that both phases are due to primary reduction steps and that the enzyme preferentially abstracts the (4R)-proton. n-Octylamine, frequently present  in turnover reactions because of its activating effect on the overall turnover rate of the enzyme, has a slightly inhibitory effect on the reduction step. We also show steady state kinetic patterns determined at both 25 degrees C and 15 degrees C which substantiate the results of Poulsen and Ziegler (J. Biol. Chem. 254, 6449-6455) which were determined at 37 degrees C.
mesh_terms: D000814:Aniline Compounds / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D005182:Flavin-Adenine Dinucleotide; D007700:Kinetics; D008433:Mathematics; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: D000814:Aniline Compounds; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)