Dc. Zeldin et al., METABOLISM OF EPOXYEICOSATRIENOIC ACIDS BY CYTOSOLIC EPOXIDE HYDROLASE - SUBSTRATE STRUCTURAL DETERMINANTS OF ASYMMETRIC CATALYSIS, Archives of biochemistry and biophysics, 316(1), 1995, pp. 443-451
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.