Metabolism of epoxyeicosatrienoic acids by cytosolic epoxide hydrolase: substrate structural determinants of asymmetric catalysis.
The metabolism of cis-epoxyeicosatrienoic acids (EETs), methyl cis-epoxyeicosatrienoates, and cis-epoxyeicosanoic acids by cytosolic epoxide hydrolase was studied to identify substrate structural features important for stereoselective metabolism and chiral diol formation. 14(R), 15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET, the predominant enantiomers present endogenously in rat organs, were metabolized at substantially higher rates than their antipodes. With the exception of 8(R),9(S)-EET (Km = 41 microM), differences in enantiomer hydration rates appear to be caused by Km-independent factors since the apparent Km values for the enantiomers of 14,15-, 11,12-, and 8(S),9(R)-EET were similar (between 3 and 5 microM). Chiral analysis of the diols resulting from enzymatic hydration of homochiral EETs showed that the regio and/or stereochemistry of water addition was EET regioisomer dependent. For the 11,12-EET enantiomers, water addition was nonregioselective; whereas, with both 8,9-EET antipodes water addition occurred predominantly at C9. Importantly, for 14,15-EET the regiochemistry of water addition was enantiomer-dependent. Only with 14(R),15(S)-EET did enzymatic hydration result in regiospecific addition at C15. Hence, enantioselective EET hydration is determined, principally, by enantiomer specific differences in rates of catalytic turnover and/or substrate binding parameters. On the other hand, the chirality of the diol products is determined by EET enantiomer-dependent differences in the regiochemistry of enzymatic oxirane cleavage and water addition. Esterification resulted in an overall reduction in the rates of epoxide hydration for all three EET-methyl esters (59, 89, and 68% of the EET rate for 8,9-, 11,12-, and 14,15-EET-methyl ester, respectively) and in the loss of regioselectivity during methyl 8(S),9(R)-EET oxirane cleavage. Catalytic EET hydrogenation reduced the rates of EET hydration (56, 45, and 23% of the EET rates for 8,9-, 11,12-, and 14,15-epoxyeicosanoic acids, respectively). Compared to 14,15-EET, enzyme catalyzed hydration of 14,15-epoxyeicosanoic acid was less regioselective and yielded products with a substantially lower chiral purity. Based on these data, as well as on the documentation of 14(R),15(R)-dihydroxyeicosatrienoic acid as an endogenous constituent of rat urine we concluded that: (1) cytosolic epoxide hydrolase plays a significant role in the regio- and stereoselective metabolism of endogenous EETs; (2) differences in the affinities and/or turnover rates of the enzyme for the individual EET antipodes may be responsible for enantioselective EET metabolism; and (3) for 14,15- and 8,9-EET, regioselective and/or enantioselective oxirane water addition is responsible for asymmetric diol formation.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Medicine, Vanderbilt University Medical School, Nashville, Tennessee 37236.
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Zeldin
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Wei
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Falck
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Hammock
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Snapper
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Capdevila
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology; D004851:Epoxide Hydrolases / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism* / Q000652:urine; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D013237:Stereoisomerism; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
D004852:Epoxy Compounds; D005231:Fatty Acids, Unsaturated; D014867:Water; D004851:Epoxide Hydrolases; D015126:8,11,14-Eicosatrienoic Acid