Leukotriene A4 hydrolase, a bifunctional enzyme Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site‐directed mutagenesis at Glu‐297

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Abstract
We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu‐297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His‐296, His‐300 and Glu‐319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site‐directed mutagenesis experiments were carried out. Glu‐297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu‐297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.