Methionine S-oxidation in human and rabbit liver microsomes: evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3.
Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.
Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, 53706, USA.
S L
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Ripp
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Itagaki
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Philpot
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Elfarra
D000328:Adult; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005260:Female; D005415:Flavins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D005415:Flavins; D011994:Recombinant Proteins; C065299:S-allylcysteine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D003545:Cysteine