Analysis of the molecular mechanism of substrate-mediated inactivation of leukotriene A4 hydrolase.
The bifunctional leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4. During exposure to the substrate leukotriene A4, a labile allylic epoxide, the enzyme is gradually inactivated as a consequence of the covalent binding of leukotriene A4 to the active site. This phenomenon, commonly referred to as suicide inactivation, has previously been rationalized as a mechanism-based process in which the enzyme converts the substrate to a highly reactive intermediate within an activated enzyme-substrate complex that partitions between covalent bond formation (inactivation) and catalysis. To further explore the molecular mechanism of the self-inactivation of leukotriene A4 hydrolase by leukotriene A4, we prepared and analyzed mutated forms of the enzyme that were either catalytically incompetent or fully active but resistant toward substrate-mediated inactivation. These mutants were treated with leukotriene A4 and leukotriene A4 methyl and ethyl esters and subjected to differential peptide mapping and enzyme activity determinations, which showed that inactivation and/or covalent modification can be completely dissociated from catalysis. Our results, together with recent findings described in the literature, argue against a mechanism-based model for suicide inactivation. We conclude that the collected data on the substrate-mediated inactivation of leukotriene A4 hydrolase best conforms to an affinity-labeling mechanism.
Mueller
M J
MJ
Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Andberg
M
M
Haeggström
J Z
JZ
D000345:Affinity Labels; D001665:Binding Sites; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors*; D004852:Epoxy Compounds / Q000737:chemistry; D006801:Humans; D017572:Leukotriene A4 / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011485:Protein Binding; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D004852:Epoxy Compounds; D017572:Leukotriene A4; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase