Kinetic mechanism of the enantioselective conversion of styrene oxide by epoxide hydrolase from Agrobacterium radiobacter AD1

Epoxide hydrolase from Agrobacterium radiobacter ADI catalyzes the enantios elective hydrolysis of styrene oxide with an E value of 16. The (R)-enantio mer of styrene oxide is first converted with a k(cat) of 3.8 s(-1), and the conversion of the (S)-enantiomer is inhibited. The latter is subsequently hydrolyzed with a k(cat) of 10.5 s(-1). The pre-steady-state kinetic parame ters were determined for both enantiomers with stopped-flow fluorescence an d rapid-quench techniques. For (R)-styrene oxide a four-step mechanism was needed to describe the data. It involved the formation of a Michaelis compl ex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme. A unimolecular isomerizatio n of the alkylated enzyme precedes the hydrolysis of the covalent intermedi ate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step. The conversion of (S)-styrene oxide could b e described by a three-step mechanism, which also involved reversible and r apid formation of an eater intermediate from a Michaelis complex and its su bsequent slow hydrolysis as the rate-limiting step. The unimolecular isomer ization step has not been observed for rat microsomal epoxide hydrolase, fo r which a kinetic mechanism was recently established [Tzeng, H.-F., Laughli n, L. T., Lin, S., and Armstrong, R. N. (1996) J. Am. Chem. SOC, 118, 9436- 9437]. For both enantiomers of styrene oxide, the K-m value was much lower than the substrate binding constant Ks due to extensive accumulation of the covalent intermediate. The enantioselectivity was more pronounced in the a lkylation rates than in the rate-limiting hydrolysis steps. The combined re action schemes for (R)- and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene ox ide.