Shape and specificity in mammalian 15-lipoxygenase active site - The functional, interplay of sequence determinants for the reaction specificity

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that t he size of the substrate binding pocket may play an essential role in deter mining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases, Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site i n 15-lipoxygenases, We found that substitution of Ile593 with alanine shift ed the positional specificity of this enzyme toward 12-lipoxygenation, To c ompare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or larg e amino acids (phenylalanine-scan) at critical positions surrounding the pu tative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density withi n the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15 -lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for Lipoxygenation spec ificity. If the 15-lipoxygenase active site is enlarged excessively, howeve r, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid bindin g pocket in defining the specificity of lipoxygenase reaction.