Change in the positional specificity of lipoxygenase 1 due to insertion of fatty acids into phosphatidylcholine deoxycholate mixed micelles.
Linoleic and arachidonic acids were inserted into phosphatidylcholine deoxycholate mixed micelles (PDM-micelles) with their tail groups buried inside and carboxylic groups exposed outside. The fatty acid hydrophobic tail had a high affinity for the hydrophobic region of phosphatidylcholine micelles. The fatty acids inserted into phosphatidylcholine micelles were better substrates for soybean lipoxygenase 1 (LOX1) with two distinct pH optima at 7.0 and 10.0. With Tween 20-solubilized linoleic acid, the enzyme had a pH optimum at 9.0, exclusively forming 13-hydroperoxides. However, with linoleic and arachidonic acids inserted into PDM-micelles, LOX1 synthesized exclusively 9- and 5-hydroperoxides, respectively. The enzyme brought about the transformation of the substrate either at pH 7.4 or at 10.0, less efficiently at pH 10.0. However, the regioselectivity of the enzyme was not altered by increasing the pH from 7.4 to 10.0. Thus, LOX1 could utilize fatty acids bound to membranes as physiological substrates. The enzyme utilized the carboxylic group of linoleic and arachidonic acids inserted into the PDM-micelles as a recognition site to convert the compounds into 9- and 5-hydroperoxides, respectively. This was confirmed by activity measurements using methyl linoleate as the substrate. Circular dichroism measurement of LOX1 with PDM-micelles suggested that while there was a small change in the tertiary structure of LOX1, the secondary structure was unaffected. Soybean LOX1, which is arachidonate 15-LOX, acted as "5-LOX", thus making it possible to change the regiospecificity of the LOX1-catalyzed reaction by altering the physical state of the substrate.
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Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, India.
Sudharshan
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Appu Rao
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D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D003840:Deoxycholic Acid / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D008823:Micelles; D008856:Microscopy, Fluorescence; D009994:Osmolar Concentration; D010100:Oxygen / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology; D013025:Glycine max / Q000201:enzymology; D013379:Substrate Specificity
D000817:Anilino Naphthalenesulfonates; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008024:Ligands; D008823:Micelles; D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; C027132:1-anilino-8-naphthalenesulfonate; D008084:Lipoxygenase; D010100:Oxygen