Positional- and stereo-selectivity of fatty acid oxygenation catalysed by mouse (12S)-lipoxygenase isoenzymes

A quantitative stereochemical analysis of the products generated by recombi nant mouse (12S)-lipoxygenase isoenzymes was performed with arachidonic aci d and linoleic acid as substrates. The leucocyte-type (12S)-lipoxygenase ge nerated, in addition to 12-hydroxyeicosatetraenoic acid (12-HETE) as the ma in product, 15- and 8-HETE from arachidonic acid and 13- and 9-hydroxyoctad ecadienoic acid (13- and 9-HODE) from linoleic acid. The platelet-type enzy me oxygenated arachidonic acid to 12- and 8-HETE and linoleic acid to 13- a nd 9-HODE, whereas the epidermis-type (12S)-lipoxygenase reaction was essen tially mono-specific with arachidonic acid but oxygenated linoleic acid to both 13- and 9-HODE. 12-HETE and 13-HODE were almost exclusively the S enan tiomers. 8-METE was the R enantiomer as a side-product of the platelet-type (12S)-lipoxygenase reaction but the S enantiomer as a side-product of the leucocyte-type reaction. 9-HODE was generated as the R enantiomer by the pl atelet-type and the epidermis-type isoenzymes and as the S enantiomer by th e leucocyte-type (12S)-lipoxygenase. On the basis of published models of li poxygenase-substrate interaction, the stereochemistry of the products gener ated by the platelet- and epidermis-type (12S)-lipoxygenases is in agreemen t with a fixed 'tail-to-head' orientation of the substrate fatty acid in th e binding pocket of these enzymes, whereas that of the reaction products of the leucocyte-type (12S)-lipoxygenase can be explained only when the inver se orientation of the substrate or a rotational isomerism along the longitu dinal axis of the substrate is allowed. Both the product spectra generated and the sensitivity towards the 12-lipoxygenase selective inhibitors N-benz yl-N-hydroxy-4-phenylpentanamide and cinnamyl-3,4-dihydroxy-alpha-cyanocinn amate indicated the platelet-type and the epidermis-type isoenzymes to be b iochemically more related to each other than to the leucocyte-type (12S)-li poxygenase.