Investigation of the mechanism of non-turnover-dependent inactivation of purified human 5-lipoxygenase. Inactivation by H2O2 and inhibition by metal ions.
Human 5-lipoxygenase is a non-heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non-turnover-dependent inactivation of purified recombinant human 5-lipoxygenase for the following reasons: catalase protects against oxygen-dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 microM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton-type reaction of the ferrous enzyme with H2O2, resulting in the formation of an activated oxygen species. Purification of 5-lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 mumol 5(S)-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5-lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half-maximal product formation being obtained at 0.9 microM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5-Lipoxygenase is strongly inhibited by low concentrations (< or = 10 microM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2 = 2 min) in the presence of excess EDTA. These observations on the mechanism of non-turnover-dependent inactivation of 5-lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.
Percival
M D
MD
Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, Pointe-Claire, Dorval, Canada.
Denis
D
D
Riendeau
D
D
Gresser
M J
MJ
D001094:Arachidonate 5-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002413:Cations, Divalent; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D015289:Leukotrienes / Q000096:biosynthesis; D016859:Lipoxygenase Inhibitors; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D019159:Nonheme Iron Proteins; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D008667:Metalloproteins; D008670:Metals; D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; C025348:arachidonic acid 5-hydroperoxide; D006861:Hydrogen Peroxide; D002374:Catalase; D001094:Arachidonate 5-Lipoxygenase; D004229:Dithiothreitol