Soybean epoxide hydrolase: identification of the catalytic residues and probing of the reaction mechanism with secondary kinetic isotope effects.
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 His(320), 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 Asp(126) 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 His(320) in the "charge relay system," mutation of Asp(285) 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 Glu(195), another acidic residue of the active site. Our studies have also confirmed the fundamental role of the conserved Tyr(175) and Tyr(255) 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/K(m)) approximately 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 Asp(126) takes place.
Blée
Elizabeth
E
Laboratoire des Phytooxylipines, IBMP-CNRS-UPR 2357, 28-Rue Goethe, Strasbourg, 67083 Cedex, France.
Summerer
Stephan
S
Flenet
Martine
M
Rogniaux
Hélène
H
Van Dorsselaer
Alain
A
Schuber
Francis
F
D020134:Catalytic Domain; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013025:Glycine max / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization
D004851:Epoxide Hydrolases