Kinetic studies on the inactivation of 5-lipoxygenase by 5(S)-hydroperoxyeicosatetraenoic acid.
The oxygenation of arachidonic acid (AA) by guinea-pig neutrophil 5-lipoxygenase terminates prematurely at a substrate utilization of only 50%. In the presence of dithiothreitol (DTT), reaction progress continues longer but still terminates prematurely, at about 70% substrate turnover. The addition of more substrate during the first 60 seconds of the initial reaction resulted in continued product formation. However, at times after 120 seconds, the addition of more AA could not produce additional product formation. Together, these results indicate a time-dependent (t1/2 = 0.5-1.0 min), irreversible loss of enzyme activity. To determine if the product 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) mediates the inactivation, it was tested for its ability to irreversibly inhibit the enzyme and found to inactivate 5-lipoxygenase with Ki = 0.05 +/- 0.01 microM and ki = 1.4 +/- 0.4 min-1. DTT changed the apparent affinity of 5-HPETE (Ki = 0.33 +/- 0.09 microM) but had no effect on the rate of inactivation (ki = 1.26 +/- 0.62 min-1). In contrast, the hydroxy derivative of 5-HPETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), is a reversible, time-independent inhibitor with Ki = 6.3 +/- 0.9 microM regardless of DTT. The ability of thiols to protect 5-lipoxygenase from production inactivation is due, at least in part, to a non-enzymatic reaction between DTT and 5-HPETE that converts the hydroperoxy acid to a material that can no longer inactivate the enzyme.
D000818:Animals; D001091:Arachidonate Lipoxygenases / Q000037:antagonists & inhibitors*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D001095:Arachidonic Acids; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D013438:Sulfhydryl Compounds; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D001091:Arachidonate Lipoxygenases; D004229:Dithiothreitol