Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity.
Kinetic studies on mechanism of the flavin-containing monooxygenase (FMO1) from pig liver microsomes are described in detail with special emphasis on the interpretation of constants derived from the rate equation. The evidence reviewed indicates that oxidation of xenobiotic substrates by the 4a-hydroperoxyflavin form of the enzyme is a second order reaction not saturable by substrate. Under steady-state conditions decomposition of the hydroxyflavin (an intermediate form of the enzyme that does not require enzyme-substrate or enzyme-product equilibrium complexes) is rate limiting. The lack of detectable equilibrium binding is also consistent with rate constants defining Km deduced from steady-state measurements. A model consistent with all evidence currently available indicates that at saturating concentrations of xenobiotic substrates that catalytic site on the enzyme is unoccupied most of the time. This property may explain why non-substrate analogs of xenobiotic substrates do not inhibit FMO activity. Rate constants for the oxidation of xenobiotics by the enzyme-bound and synthetic 4a-hydroperoxyflavin indicate that while enzyme protein accelerates the reaction with xenobiotics bearing nitrogen, it has only marginal effects on the oxidation of substrates bearing sulfur. Differences in the nucleophilicity of compounds bearing these heteroatoms may be primarily responsible but other, as yet undefined, factors may also contribute. In addition, analysis of rate constants affected by protonated lipophilic amines indicates that these allosteric effectors apparently modify enzyme structure so as to affect two or more rate constants and, depending on the nature and concentration of the xenobiotic substrate, protonated amines can either stimulate or inhibit catalytic activity.
Poulsen
L L
LL
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA.
Ziegler
D M
DM
D000588:Amines / Q000378:metabolism; D000818:Animals; D005415:Flavins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine; D015262:Xenobiotics / Q000378:metabolism
D000588:Amines; D005415:Flavins; D015262:Xenobiotics; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)