METABOLISM OF EPOXYEICOSATRIENOIC ACIDS BY CYTOSOLIC EPOXIDE HYDROLASE - SUBSTRATE STRUCTURAL DETERMINANTS OF ASYMMETRIC CATALYSIS

The metabolism of cis-epoxyeicosatrienoic acids (EETs), methyl cis-epo xyeicosatrienoates, and cis-epoxyeicosanoic acids by cytosolic epoxide hydrolase was studied to identify substrate structural features impor tant for stereoselective metabolism and chiral diol formation. 14(R), 15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET, the predominant enantiomers p resent endogenously in rat organs, were metabolized at substantially h igher rates than their antipodes. With the exception of 8(R),9(S)-EET (K-m = 41 mu M), differences in enantiomer hydration rates appear to b e caused by K-m-independent factors since the apparent K-m values for the enantiomers of 14,15-, 11,12-, and 8(S),9(R)-EET were similar (bet ween 3 and 5 mu M). Chiral analysis of the diols resulting from enzyma tic hydration of homochiral EETs showed that the regio and/or stereoch emistry 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. Im portantly, for 14,15-EET the regiochemistry of water addition was enan tiomer-dependent. Only with 14(R),15(S)-EET did enzymatic hydration re sult in regiospecific addition at C15. Hence, enantioselective EET hyd ration 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 E ET enantiomer-dependent differences in the regiochemistry of enzymatic oxirane cleavage and water addition. Esterification resulted in an ov erall reduction in the rates of epoxide hydration for all three EET-me thyl 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 hydrogena tion reduced the rates of EET hydration (56, 45, and 23% of the EET ra tes for 8,9-, 11,12-, and 14,15-epoxyeicosanoic acids, respectively). Compared to 14,15-EET, enzyme catalyzed hydration of 14,15-epoxyeicosa noic acid was less regioselective and yielded products with a substant ially lower chiral purity. Based on these data, as well as on the docu mentation of 14(R),15(R)-dihydroxyeicosatrienoic acid as an endogenous constituent of rat urine we concluded that: (1) cytosolic epoxide hyd rolase plays a significant role in the regio- and stereoselective meta bolism of endogenous EETs; (2) differences in the affinities and/or tu rnover rates of the enzyme for the individual EET antipodes may be res ponsible 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 coor dinates responsible for the asymmetry of EET hydration and diol format ion must be circumscribed by a highly structured active site capable o f recognizing, regio- and stereospecifically, overall substrate polari ty, freedom of C-C bond rotation, and/or protein-substrate pi-pi dipol e interactions. (C) 1995 Academic Press,Inc.