Metabolism of Epoxyeicosatrienoic Acids by Cytosolic Epoxide Hydrolase: Substrate Structural Determinants of Asymmetric Catalysis

D. C. Zeldin, S. Z. Wei, J. R. Falck, B. D. Hammock, J. R. Snapper, J. H. Capdevila

Research output: Contribution to journalArticle

106 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)443-451
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume316
Issue number1
DOIs
StatePublished - Jan 1995

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Epoxide Hydrolases
Enantiomers
Catalysis
Metabolism
Hydration
NSC 153174
Acids
Substrates
Ethylene Oxide
Water
Rats
Esters
Regioselectivity
Stereochemistry
Hydrogenation
8,9-epoxyeicosatrienoic acid
Esterification
Chirality
Epoxy Compounds
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Metabolism of Epoxyeicosatrienoic Acids by Cytosolic Epoxide Hydrolase : Substrate Structural Determinants of Asymmetric Catalysis. / Zeldin, D. C.; Wei, S. Z.; Falck, J. R.; Hammock, B. D.; Snapper, J. R.; Capdevila, J. H.

In: Archives of Biochemistry and Biophysics, Vol. 316, No. 1, 01.1995, p. 443-451.

Research output: Contribution to journalArticle

Zeldin, D. C. ; Wei, S. Z. ; Falck, J. R. ; Hammock, B. D. ; Snapper, J. R. ; Capdevila, J. H. / Metabolism of Epoxyeicosatrienoic Acids by Cytosolic Epoxide Hydrolase : Substrate Structural Determinants of Asymmetric Catalysis. In: Archives of Biochemistry and Biophysics. 1995 ; Vol. 316, No. 1. pp. 443-451.
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abstract = "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.",
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T1 - Metabolism of Epoxyeicosatrienoic Acids by Cytosolic Epoxide Hydrolase

T2 - Substrate Structural Determinants of Asymmetric Catalysis

AU - Zeldin, D. C.

AU - Wei, S. Z.

AU - Falck, J. R.

AU - Hammock, B. D.

AU - Snapper, J. R.

AU - Capdevila, J. H.

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N2 - 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.

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