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

Peter C. Rudberg, Fredrik Tholander, Martina Andberg, Marjolein M.G.M. Thunnissen, Jesper Z. Haeggström

Research output: Contribution to journalArticleScientificpeer-review

43 Citations (Scopus)

Abstract

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 Ki 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 (Vmax = 0.3-20%), whereas mutations of Lys565 have limited effect on catalysis (Vmax = 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.

Original languageEnglish
Pages (from-to)27376-27382
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number26
DOIs
Publication statusPublished - 25 Jun 2004
MoE publication typeA1 Journal article-refereed

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