ANALYSIS OF THE MOLECULAR MECHANISM OF SUBSTRATE-MEDIATED INACTIVATION OF LEUKOTRIENE A(4) HYDROLASE

The bifunctional leukotriene A(4) hydrolase catalyzes the final step i n the biosynthesis of the proinflammatory leukotriene B-4, During expo sure to the substrate leukotriene A(4), a labile allylic epoxide, the enzyme is gradually inactivated as a consequence of the covalent bindi ng of leukotriene A(4) to the active site. This phenomenon, commonly r eferred to as suicide inactivation, has previously been rationalized a s a mechanism-based process in which the enzyme converts the substrate to a highly reactive intermediate within an activated enzyme-substrat e complex that partitions between covalent bond formation (inactivatio n) and catalysis. To further explore the molecular mechanism of the se lf-inactivation of leukotriene A(4) hydrolase by leukotriene A(4), we prepared and analyzed mutated forms of the enzyme that were either cat alytically incompetent or fully active but resistant toward substrate- mediated inactivation. These mutants were treated with leukotriene A(4 ) and leukotriene A(4) methyl and ethyl esters and subjected to differ ential peptide mapping and enzyme activity determinations, which showe d that inactivation and/or covalent modification can be completely dis sociated from catalysis. Our results, together with recent findings de scribed in the literature, argue against a mechanism-based model for s uicide inactivation. We conclude that the collected data on the substr ate-mediated inactivation of leukotriene A(4) hydrolase best conforms to an affinity-labeling mechanism.