Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis o f epoxides to their diols via an alkyl-enzyme intermediate. The recently so lved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way t hat they can serve as proton donor in the alkylation reaction step. The rol e of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) v alues were only 2-4-fold lower than for wild-type enzyme, whereas the K-m v alues for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide wer e 3 orders of magnitude higher than the K-m values of wildtype enzyme, show ing that the alkylation half-reaction is severely affected by the mutations . Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y 215F and Y215A mutants showed that the 1000-fold elevated K-m values were m ainly caused by a 15-40-fold increase in K-S and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermedia tes were not significantly affected by the mutations. The double mutant Y15 2F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K-m value that was 6 orders of magnitude lower than with wild-type e nzyme and 3 orders of magnitude lower than with the single tyrosine mutants . This indicates that the effects of the mutations were cumulative. The sid e chain of Gln134 is positioned in the active site of the X-ray structure o f epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-typ e enzyme, indicating that Gln134 is not essential for catalysis and that th e side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resul ting in the identification of phenylacetamide as a competitive inhibitor wi th an inhibition constant of 30 mu M.