Skaterna T. Molecular mechanisms of lipoxygenase regulation in modeling systems

Enzymatic oxidation of polyunsaturated fatty acids on catalytic action lipoxygenases. Investigation of the molecular mechanisms of influence for natural compounds and synthetic derivatives of linoleic acid on the activity of lipoxygenase in systems modeling enzyme cell membrane environment. Chromatography, electrophoresis, physics parameters mixed micelle linoleic acid/Lubrol РХ; UV-spectrophotometers, kinetic, mathematic methods. Detailed study of phosphatidic acid influence on lipoxygenase function was carried out. Phosphatidic acid was shown to act as an allosteric activator of 5-lipoxygenase in micellar system, which led to the appearance of additional pH optimum in acidic region. Under conditions of nonsaturating substrate (linoleic acid ) amounts and nonphysiological pH, phosphatidic acid was proved to maintain certain level of 5-lipoxygenase mediated oxygenation of primary products by replacement of the substrate molecules in the enzyme allosteric center, increasing in such way enzyme affinity to substrate and decreasing the part of nonenzyme fraction products of linoleic acid oxygenation.Thermodynamic parameters of 5-lipoxygenase thermoinactivation were determined. Thermoinactivation rate constants and activation energy of enzyme thermoinactivation were shown to increase in the presence of phosphatidic acid. It was suggested that hydrophobic forces play an essential role for 5-lipoxygenase and phosphatidic acid interaction, that can induces certain conformational changes of enzyme molecule.The model system with increased quantity of mixed micelles (with constant molar ratio of nonionic detergent Lubrol PX and substrate - linoleic acid) was developed and optimized for studies of membrane binding enzymes. Using such model system we found that linoleyl hydroxamic acid acted as a noncompetitive inhibitor of 5-lipoxygenase mediated linoleic acid oxygenation. It was also shown that linoleyl hydroxamic acid oxidized derivatives exert the same inhibition effects as nonoxidized linoleyl hydroxamic acid on plant and animal lipoxygenases, namely potato tuber 5-lipoxygenase and porcine leucocyte 12-lipoxygenase. Sphere of the use is bioorganic chemistry, molecular biology.