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Catalytic triad of microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a strongly increased turnover rate


Arand, M; Müller, F; Mecky, A; Hinz, W; Urban, P; Pompon, D; Kellner, R; Oesch, F (1999). Catalytic triad of microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a strongly increased turnover rate. Biochemical Journal, 337 ( Pt:37-43.

Abstract

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

Abstract

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Pharmacology and Toxicology
07 Faculty of Science > Institute of Pharmacology and Toxicology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:1 January 1999
Deposited On:29 Oct 2015 13:38
Last Modified:05 Apr 2016 19:29
Publisher:Portland Press
ISSN:0264-6021
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1042/0264-6021:3370037
PubMed ID:9854022

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