Abstract
Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining 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 in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within 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 specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.
Highlights
Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5, 12, and 15-lipoxygenases
Amino Acid 593 Is Important for the Positional Specificity of 12/15-LOs—The crystal structure of the rabbit 15-LO predicted that Ile593, sitting near the base of the substrate binding pocket, may play a role in the alignment of arachidonic acid at the active site and may influence the positional specificity of the enzyme [15]
We found that the major reaction products of arachidonic acid oxygenation by the I593A mutant, 15S-H(P)ETE and 12S-H(P)ETE were formed in a ratio of 57/43, compared with 97/3 for the wild-type enzyme (Fig. 1)
Summary
Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Enlargement or alteration in packing density within 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 specificity. If the 15-lipoxygenase active site is enlarged excessively, no lipoxygenation was observed anymore Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction. Molecular modeling of critical residues that differ among various LO isoforms using the threedimensional structure of the rabbit 15-LO, suggested that the volume at the base of the substrate binding pocket could be important for the positional specificity. To examine the importance of the pocket volume in determining the specificity of oxygenation, we added or deleted mass at the bottom of the pocket in 15-LO to test whether such
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
