Implications for an ionized alkyl-enzyme intermediate during StEH1-catalyzed trans-stilbene oxide hydrolysis.

The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.