DEFINING THE ARACHIDONIC-ACID BINDING-SITE OF HUMAN 15-LIPOXYGENASE -MOLECULAR MODELING AND MUTAGENESIS

Mammalian lipoxygenases have been implicated in the pathogenesis of se veral inflammatory disorders and are, therefore, important targets for drug discovery, Both plant and mammalian lipoxygenases catalyze the d ioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lip oxygenase for which an atomic resolution structure had been determined , No structure of lipoxygenase with substrate or inhibitor bound is cu rrently available, A model of arachidonic acid docked into the propose d substrate binding site in the soybean structure is presented here. A nalysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding, Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity be tween the soybean and mammalian enzymes, Both a positively charged ami no acid residue (Arg(402)) and an aromatic amino acid residue (Phe(414 )) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be char acteristic of non-enzymatic fatty acid-binding proteins are now implic ated in the substrate binding pocket of lipoxygenases.