SITE-DIRECTED MUTAGENESIS STUDIES ON THE IRON-BINDING DOMAIN AND THE DETERMINANT FOR THE SUBSTRATE OXYGENATION SITE OF PORCINE LEUKOCYTE ARACHIDONATE 12-LIPOXYGENASE

cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expres sed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific a ctivity of about 1.5 mu mol/min per mg protein. Each of eight histidin e residues, which were well-conserved among various mammalian lipoxyge nases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaff inity-purified to near homogeneity. Mutations of His-361, -366 and -54 1 caused a total loss of enzyme activity, and the iron content was muc h lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild -type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased th e activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precis ely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12- lipoxygenase. Since a high homology of amino acid sequence was known b etween porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenase s, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the rati o of 15- and 12-lipoxygenase activities from 0.1 to 5.7.