Saccharomyces cerevisiae Leukotriene A₄ Hydrolase:  Formation of Leukotriene B₄ and Identification of Catalytic Residues^†

Leukotriene A₄ hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A₄ into the proinflammatory mediator leukotriene B₄, and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A₄ hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A₄ hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A₄ into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B₄, and Δ⁶-trans-Δ⁸-cis-leukotriene B₄, with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A₄ hydrolase to leukotriene A₄ selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A₄ hydrolase suggest that leukotriene A₄ binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A₄ hydrolase for a Tyr, the corresponding residue in human leukotriene A₄ hydrolase, results in a protein that converts leukotriene A₄ into leukotriene B₄ with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A₄, thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A₄ hydrolase toward its mammalian counterparts.