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

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.

Epoxide hydrolases: biochemistry and molecular biology

“…Like sEH, mEH is a member of the a/b-hydrolase enzyme family [66]. Based on its similarity with bacterial haloalkane dehalogenases and other epoxide hydrolases, a catalytic triad consisting of a His 431 , Asp 226 , and a Glu 404 has been proposed [129,130]. These findings are based on earlier reports of a His at position 431 that is essential for catalysis [131], and sequence alignments of related epoxide hydrolases.…”

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

“…hydrolases generally resulted in inactive proteins, consistent with the role of this residue as an essential catalytic base (4,9,17,24,35,44,53,56,65). However, reports on protein variants of C-C hydrolase MhpC (40) and Agrobacterium radiobacter epoxide hydrolase (59) show that the observation of minor residual activity after replacement of the triad's histidine is not unprecedented.…”

Detoxication Strategy of Epoxide Hydrolase—The Basis for a Novel Threshold for Definable Genotoxic Carcinogens

“…Armstrong (1999) calculated, for glycidyl-4-nitrobenzoate as the substrate, the rate constant of step 1 to be three orders of magnitude higher than the rate constant for step 2 (Tzeng et al, 1996). According to our own work, similar reaction kinetics exist for the turnover of 9,10-epoxystearic acid (Arand et al, 1999) and styrene oxide. The most important point from this new insight in the enzymatic mechanism of mEH is to realize that the rate of product formation does not adequately mirror the detoxification efficiency of the enzyme.…”

“…There is a fair number of direct-acting mutagens/carcinogens, but most genotoxic agents require metabolic activation (Oesch and Arand, 1999). To allow elimination of initially lipophilic compounds via the aqueous excretion systems of the mammalian body, a huge network of xenobiotic-metabolizing enzymes has evolved.…”