Catalytic triad of microsomal epoxide hydrolase:: replacement of Glu404 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 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 Asp(226), Glu(404) and His(431). 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 Glu(404) and His(431) 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 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 intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp(226). Surprisingly, the replacement of Glu(404) with Asp greatly increased the V-max of the enzyme with styrene 7,8-oxide (23-fold) and 9,10-epoxystearic acid (39-fold). The increase 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 the enzymic reaction. Owing to its enhanced catalytic properties, the Glu(404) --> 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.