Fig. 3.
Epoxide hydrolysis by recombinant microsomal and soluble EHs. (A) Epoxide hydrolysis of oprozomib by recombinant human mEH and sEH. Formation of PR-176 was quantified after incubation of oprozomib (2 µM) with various concentrations of enzymes for 30 minutes. A pilot study has shown a linear formation of PR-176 within 30 minutes under the current conditions. (B) Epoxide hydrolysis of cis-SO and trans-SO (50 µM) by mEH and sEH (5 µg/ml), respectively. Significant diol product formation was observed after incubation for 10 or 20 minutes, confirming the hydrolysis activities of EHs. (C) Kinetics of the formation of PR-176 using recombinant mEH. A range of oprozomib concentrations were incubated with mEH (2 µg/ml) at 37°C for 30 minutes. (D) Kinetics of cis-SO hydrolysis using recombinant mEH. A series of cis-SO concentrations were incubated with mEH (4 μg/ml) at 37°C for 10 minutes. Data represent mean ± S.D. from three replicate incubations. The Km and Vmax values were estimated by fitting the curve into the Michaelis-Menten equation.