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Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli.
Arch Biochem Biophys. 1994 Jun; 311(2):369-77.AB

Abstract

Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.

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Authors+Show Affiliations

Rettie AE
Department of Medicinal Chemistry, University of Washington, Seattle 98195.
Lawton MP
No affiliation info available
Sadeque AJ
No affiliation info available
Meier GP
No affiliation info available
Philpot RM
No affiliation info available

MeSH

AlkylationAnimalsCloning, MolecularDetergentsEscherichia coliIsoenzymesKineticsMicrosomes, LiverOxygenasesRabbitsRecombinant ProteinsSubstrate SpecificitySulfides

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8203899

Citation

Rettie, A E., et al. "Prochiral Sulfoxidation as a Probe for Multiple Forms of the Microsomal Flavin-containing Monooxygenase: Studies With Rabbit FMO1, FMO2, FMO3, and FMO5 Expressed in Escherichia Coli." Archives of Biochemistry and Biophysics, vol. 311, no. 2, 1994, pp. 369-77.
Rettie AE, Lawton MP, Sadeque AJ, et al. Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli. Arch Biochem Biophys. 1994;311(2):369-77.
Rettie, A. E., Lawton, M. P., Sadeque, A. J., Meier, G. P., & Philpot, R. M. (1994). Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli. Archives of Biochemistry and Biophysics, 311(2), 369-77.
Rettie AE, et al. Prochiral Sulfoxidation as a Probe for Multiple Forms of the Microsomal Flavin-containing Monooxygenase: Studies With Rabbit FMO1, FMO2, FMO3, and FMO5 Expressed in Escherichia Coli. Arch Biochem Biophys. 1994;311(2):369-77. PubMed PMID: 8203899.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli. AU - Rettie,A E, AU - Lawton,M P, AU - Sadeque,A J, AU - Meier,G P, AU - Philpot,R M, PY - 1994/6/1/pubmed PY - 1994/6/1/medline PY - 1994/6/1/entrez SP - 369 EP - 77 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 311 IS - 2 N2 - Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/8203899/Prochiral_sulfoxidation_as_a_probe_for_multiple_forms_of_the_microsomal_flavin_containing_monooxygenase:_studies_with_rabbit_FMO1_FMO2_FMO3_and_FMO5_expressed_in_Escherichia_coli_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(84)71250-1 DB - PRIME DP - Unbound Medicine ER -