Abstract
Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr465 and Tyr381 act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr465 and Tyr381 are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr465 and Tyr381 decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr465 is conserved in microsomal EHs. The substitution of analogous Tyr374 with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 23082-23088 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 275 |
| Issue number | 30 |
| DOIs | |
| State | Published - Jul 28 2000 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase. / Yamada, Takashi; Morisseau, Christophe; Maxwell, Joseph E.; Argiriadi, Maria A.; Christianson, David W.; Hammock, Bruce D.
In: Journal of Biological Chemistry, Vol. 275, No. 30, 28.07.2000, p. 23082-23088.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase
AU - Yamada, Takashi
AU - Morisseau, Christophe
AU - Maxwell, Joseph E.
AU - Argiriadi, Maria A.
AU - Christianson, David W.
AU - Hammock, Bruce D.
PY - 2000/7/28
Y1 - 2000/7/28
N2 - Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr465 and Tyr381 act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr465 and Tyr381 are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr465 and Tyr381 decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr465 is conserved in microsomal EHs. The substitution of analogous Tyr374 with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.
AB - Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr465 and Tyr381 act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr465 and Tyr381 are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr465 and Tyr381 decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr465 is conserved in microsomal EHs. The substitution of analogous Tyr374 with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.
UR - http://www.scopus.com/inward/record.url?scp=0034725581&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034725581&partnerID=8YFLogxK
U2 - 10.1074/jbc.M001464200
DO - 10.1074/jbc.M001464200
M3 - Article
C2 - 10806198
AN - SCOPUS:0034725581
VL - 275
SP - 23082
EP - 23088
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 30
ER -