title: Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate.
abstract: Epoxide hydrolases catalyze hydrolytic epoxide ring-opening, most often via formation of a covalent hydroxyalkyl-enzyme intermediate. A mutant of Agrobacterium radiobacter epoxide hydrolase, in which the phenylalanine residue that flanks the invariant catalytic aspartate nucleophile is replaced by a threonine, exhibited inactivation during conversion when the (R)-enantiomer of para-nitrostyrene epoxide was used as substrate. HPLC analysis of tryptic fragments of the epoxide hydrolase, followed by MALDI-TOF and TOF/TOF analysis, indicated that inactivation was due to conversion of the nucleophilic aspartate into isoaspartate, which represents a novel mechanism of catalysis-induced autoinactivation. Inactivation occurred at a lower rate with the (S)-enantiomer of para-nitrostyrene epoxide, indicating that it is related to the structure of the covalent hydroxyalkyl-enzyme intermediate.
authors.affiliation: Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Bert
authors.initials: B
authors.lastname: van Loo
authors.forename: Hjalmar P
authors.initials: HP
authors.lastname: Permentier
authors.forename: Jaap
authors.initials: J
authors.lastname: Kingma
authors.forename: Helen
authors.initials: H
authors.lastname: Baldascini
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D004852:Epoxy Compounds / Q000737:chemistry*; D026581:Isoaspartic Acid / Q000737:chemistry*; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004852:Epoxy Compounds; D026581:Isoaspartic Acid; D013912:Threonine; D001224:Aspartic Acid; D010649:Phenylalanine; C033332:4-nitrostyrene oxide; D004851:Epoxide Hydrolases