Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the micros omal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 rev ealed that the enzyme is inactivated through modification of Asp/Glu and Hi s residues, as well as through modification of Ser. Since Asp acts as the n ucleophile, and Asp/Glu and His serve as charge relay partners in the catal ytic triad of microsomal and soluble epoxide hydrolases during epoxide hydr olysis, inactivation of the enzyme by modification of the Asp/Glu and His r esidues agrees with the established reaction mechanism of these enzymes. Ho wever, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the relate d L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes . Co2+, Hg2+, Ag+, Mg2+ and Ca2+ inhibited enzyme activity and EDTA increas ed enzyme activity. The activation energy for inactivation of the enzyme wa s 167 kJ mol(-1). Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on subst rate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interfac e, analogous to lipases.