Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate

van Loo, Bert; Permentier, Hjalmar P.; Kingma, Jaap; Baldascini, Helen; Janssen, Dick B.

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.

ENZYME analysis; AGROBACTERIUM; AMINO acids; AMINO compounds

FEBS Letters, 2008, Vol 582, Issue 11, p1581

0014-5793

Academic Journal

10.1016/j.febslet.2008.04.001