Leukotriene A4 hydrolase: Identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates

Leukotriene (LT) A₄ hydrolase is a bifunctional zinc metalloenzyme, which converts LTA₄ into the neutrophil chemoattractant LTB⁴ and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA₄ hydrolase, Arg⁵⁶³ and Lys⁵⁶⁵ are found at the entrance of the active center. Here we report that replacement of Arg⁵⁶³, but not Lys⁵⁶⁵, leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg⁵⁶³ do not seem to affect substrate binding strength, because values of K_i for LTA₄ are almost identical for wild type and (R563K)LTA₄ hydrolase. These results are supported by the 2.3-Å crystal structure of (R563A)LTA ₄ hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg⁵⁶³ reduce the catalytic activity (V_max = 0.3-20%), whereas mutations of Lys⁵⁶⁵ have limited effect on catalysis (V_max = 58-108%). However, in (K565A)- and (K565M)LTA ₄ hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K _m = 480-640%). Together, our data indicate that Arg⁵⁶³ plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg⁵⁶³ and Lys⁵⁶⁵ seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg⁵⁶³ and Lys⁵⁶⁵ possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA ₄ hydrolase.