Leukotriene A(4) hydrolase: Protection from mechanism-based inactivation by mutation of tyrosine-378

Leukotriene A(4) (LTA(4)) hydrolase [(7E,9E, 11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9,11,14-tetraenoate hydro-lase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B-4 (LTB(4)), LTA(4) hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA(4) to the protein in a 1:1 stoichiometry, Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA(4) hydrolase, which contains a site involved in the covalent binding of LTA(4) to the native enzyme, To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gin in two separate mutants, In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala, The mutated enzymes were expressed as (His)(6)-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA(4), revealed that wild-type enzyme and the mutants [S379A] and [S380A] LTA(4) hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA(4). Furthermore, in [Y378F]LTA(4) hydrolase, the value of k(cat) for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme, Thus, by a single-point mutation in LTA(4) hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.