Rat 12-lipoxygenase: mutations of amino acids implicated in the positional specificity of 15- and 12-lipoxygenases.

Site directed mutagenesis, based on the polymerase chain reaction, was carried out on a rat 12-lipoxygenase cDNA within a region that encodes several amino acids believed to be of importance for the positional specificity of 15- and 12-lipoxygenases. The following mutants were constructed to increase the 15-lipoxygenase activity of rat 12-lipoxygenase; [Leu397]12-lipoxygenase, [Ile389, Leu397]12-lipoxygenase, [Gln416]12-lipoxygenase, [Ile417]12-lipoxygenase, and [Gln416, Ile417]12-lipoxygenase. Each mutated cDNA was expressed in Escherichia coli and 12- and 15-lipoxygenase activity was assayed in the 10,000 x g supernatants of homogenized cells. When compared to wild type enzyme, none of the mutants exhibited significantly increased 15-lipoxygenase activity. Thus, considering the primary structure of wild type enzyme and our results of mutagenetic replacements, we have not found evidence indicating that amino acids in positions 416, 417, or 418 are critical for the positional specificity of rat 12-lipoxygenase.