Soybean epoxide hydrolase: identification of the catalytic residues and probing of the reaction mechanism with secondary kinetic isotope effects.

Abstract Soybean epoxide hydrolase catalyzes the oxirane ring opening of 9,10-epoxystearate via a two-step mechanism involving the formation of an alkylenzyme intermediate, which, in contrast to most epoxide hydrolases studied so far, was found to be the rate-limiting step. We have probed residues potentially involved in catalysis by site-directed mutagenesis. Mutation of His320, a residue predicted from sequence analysis to belong to the catalytic triad of the enzyme, considerably slowed down the second half-reaction. This kinetic manipulation provoked an accumulation of the reaction intermediate, which could be trapped and characterized by electrospray ionization mass spectrometry. As expected, mutation of Asp126 totally abolished the activity of the enzyme from its crucial function as nucleophile involved in the formation of the alkylenzyme. In line with its role as the partner of His320 in the “charge relay system,” mutation of Asp285 dramatically reduced the rate of catalysis. However, the mutant D285L still exhibited a very low residual activity, which, by structural analysis and mutagenesis, has been tentatively attributed to Glu195, another acidic residue of the active site. Our studies have also confirmed the fundamental role of the conserved Tyr175 and Tyr255 residues, which are believed to activate the oxirane ring. Finally, we have determined the secondary tritium kinetic isotope effects on the epoxide opening step of 9,10-epoxystearate. The large observed values, i.e. T(V/Km) ≈ 1.30, can be interpreted by the occurrence of a very late transition state in which the epoxide bond is broken before the nucleophilic attack by Asp126 takes place.