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12:08
Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
Prochiral sulfoxidation as a probe for flavin-containing monooxygenases
Catherine K Yeung1, Allan E Rettie
1Department of Medicinal Chemistry, University of Washington, Seattle, USA.
Methods in Molecular Biology (Clifton, N.J.)
|May 25, 2006
Summary
Flavin-containing monooxygenase (FMO) enzymes metabolize aryl alkyl sulfides into chiral sulfoxides. This study details methods for analyzing these sulfoxide products, aiding in the discrimination of FMO isoforms.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Asymmetric aryl alkyl sulfides are metabolized by flavin-containing monooxygenase (FMO) and cytochrome P450 enzymes.
- Metabolism yields enantiomerically enriched sulfoxide products, analyzable using chiral stationary phases.
- Sulfoxidation of p-tolyl methyl sulfide probes is isoform-dependent and species-independent for FMO discrimination.
Purpose of the Study:
- To detail experimental protocols for the stereochemical analysis of sulfoxides.
- To enable discrimination among FMO1, FMO3, and FMO5 isoforms.
- To provide methods for analyzing sulfoxides generated from various alkyl p-tolyl sulfides.
Main Methods:
- Utilizing prochiral sulfoxidation of p-tolyl methyl sulfide derivatives.
- Employing purified FMO isoforms and tissue microsomal preparations.
- Quenching cytochrome P450 activity in crude preparations to isolate FMO catalysis.
- Stereochemical analysis of resulting sulfoxide products using chiral stationary phases.
Main Results:
- Enantiomerically enriched sulfoxides are produced from aryl alkyl sulfides.
- The stereochemistry of sulfoxidation is dependent on the specific FMO isoform.
- The stereochemical outcome is species-independent across tested FMOs.
- Protocols allow for the analysis of sulfoxides from methyl, ethyl, n-propyl, and n-butyl p-tolyl sulfide.
Conclusions:
- Sulfoxidation of aryl alkyl sulfides by FMOs provides a stereochemical readout for isoform identification.
- The described methods are valuable for characterizing FMO activity and substrate specificity.
- These protocols facilitate the study of drug metabolism and enzyme kinetics involving FMOs.
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