Catalytic triad of microsomal epoxide hydrolase: replacement of Glu(404) with Asp leads to a strongly increased turnover rate

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta -hydrolase fold enzymes. A catalytic triad in the active centre of the enzy me hydrolyses the substrate molecules in a two-step reaction via the interm ediate formation of an enzyme-substrate ester, Here we show that the mEH ca talytic triad is composed of Asp(226), Glu(404) and His(431). Replacing eit her of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces ce revisiae. For Glu(404) and His(431) mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester interme diate, indicating that the lack of enzymic activity is due to an indispensa ble function of either residue in the hydrolytic step of the enzymic reacti on. The role of Asp(226) as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the C-14-labelled substrate after cleavage of the ester i ntermediate with cyanogen bromide. Sequence analysis revealed that one of t he two peptides within this sample harboured Asp(226). Surprisingly, the re placement of Glu(404) with Asp greatly increased the V-max of the enzyme wi th styrene 7,8-oxide (23-fold) and 9,10-epoxystearic acid (39-fold). The in crease in V-max was paralleled by an increase in K-m with both substrates, in line with a selective enhancement of the second, rate-limiting step of t he enzymic reaction. Owing to its enhanced catalytic properties, the Glu(40 4) --> Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all na tive mEHs analysed so far have a Glu in place of the acidic charge relay re sidue is discussed.