Characterization of the arachidonate and ATP binding sites of human 5-lipoxygenase using photoaffinity labeling and enzyme immobilization.
The arachidonic acid and the ATP binding sites of human 5-lipoxygenase were characterized using photoaffinity labeling and immobilization of the enzyme on ATP-agarose. Photoaffinity labeling of the active site of 5-lipoxygenase was achieved with a novel thiopyranoindole inhibitor containing a 4-azido-3-iodobenzenesulfonyl moiety (L-708,714). This probe was found to inhibit the activity of 5-lipoxygenase (IC50 = 0.3 microM) and to covalently label the enzyme after UV light irradiation. The labeling was inhibited by arachidonic acid, N-hydroxyurea, and dihydrobenzofuranol inhibitors which have been shown to reduce the non-heme iron center of 5-lipoxygenase. Photoaffinity labeling of 5-lipoxygenase by L-708,714 was dependent on the presence of both Ca2+ ions and phospholipids and was independent of ATP. It occurred at similar levels using native (Fe2+), oxidized (Fe3+), or H2O2-inactivated enzyme, but was abolished by heat inactivation of the enzyme. Competition of the labeling by various thiopyranoindoles and other inhibitors such as L-697,198,ZD-2138, and zileuton was found to be related to their inhibitory potency. Immobilized 5-lipoxygenase on ATP-agarose was found to be selectively eluted by adenine nucleotides (ATP > ADP > AMP) but not by solutions containing high salt concentrations, mild detergents, arachidonic acid, or inhibitors. 5-Lipoxygenase inhibitors were selectively retained on the immobilized enzyme and eluted by buffer containing arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Falgueyret
J P
JP
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval Québec, Canada.
Denis
D
D
Macdonald
D
D
Hutchinson
J H
JH
Riendeau
D
D
D000227:Adenine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D016718:Arachidonic Acid / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007211:Indoles / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D007213:Indomethacin / Q000494:pharmacology; D007462:Iodobenzenes / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D016859:Lipoxygenase Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D010743:Phospholipids / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014162:Transfection; D014466:Ultraviolet Rays
C030548:ATP-sepharose; D000227:Adenine Nucleotides; D000345:Affinity Labels; D004800:Enzymes, Immobilized; D007211:Indoles; D007462:Iodobenzenes; C096344:L 708714; D016859:Lipoxygenase Inhibitors; D010743:Phospholipids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D012685:Sepharose; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium; D006918:Hydroxyurea; D007213:Indomethacin