RGD Reference Report - Catalytic triad of microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a strongly increased turnover rate. - Rat Genome Database

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

Authors: Arand, M  Müller, F  Mecky, A  Hinz, W  Urban, P  Pompon, D  Kellner, R  Oesch, F 
Citation: Arand M, etal., Biochem J. 1999 Jan 1;337 ( Pt 1):37-43.
RGD ID: 13210555
Pubmed: PMID:9854022   (View Abstract at PubMed)
PMCID: PMC1219933   (View Article at PubMed Central)

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.



Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Ephx1Ratepoxide metabolic process involved_inIDA PMID:9854022UniProt 

Cellular Component

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Ephx1Ratintracellular membrane-bounded organelle located_inIDA PMID:9854022UniProt 
Ephx1Ratmembrane located_inIDA PMID:9854022UniProt 

Molecular Function

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Ephx1Ratcis-stilbene-oxide hydrolase activity enablesIMP PMID:9854022UniProt 
Ephx1Ratepoxide hydrolase activity enablesIMP PMID:9854022UniProt 

Objects Annotated

Genes (Rattus norvegicus)
Ephx1  (epoxide hydrolase 1)


Additional Information