Site-directed mutagenesis of leukotriene A4 hydrolase: distinction of leukotriene A4 hydrolase and aminopeptidase activities.

Abstract

Leukotriene (LT) A4 hydrolase catalyzes enzymatic hydration of LTA4 to biologically active substance, LTB4. Biochemical and immuno-histochemical studies have shown that this enzyme is ubiquitously distributed in various cells and tissues. A sequence domain of LTA4 hydrolase was found to be homologous to those of several zinc metalloproteases. Both native and recombinant enzymes were shown to possess equimolar zinc ion and aminopeptidase activity. To examine the molecular mechanism of this enzyme reaction, site-directed mutagenesis experiments were carried out. Single amino acid substitutions at Glu-297 revealed a distinction of two enzyme activities, and suggest that the glutamic acid residue at 297 is essential for aminopeptidase, while the side chain of Glu or Gln is required for LTA4 hydrolase activity. The loss of two enzyme activities in a mutant E319K confirmed the proposal that the presence of a zinc ion in the enzyme is required for both enzyme activities.