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 μM), 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 μM). 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. The protein spatial coordinates responsible for the asymmetry of EET hydration and diol formation must be circumscribed by a highly structured active site capable of recognizing, regio- and stereospecifically, overall substrate polarity, freedom of CC bond rotation, and/or protein-substrate π-π dipole interactions.
ASJC Scopus subject areas
- Molecular Biology