During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the ω-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of ω-modified fatty acids by 15-lipoxygenases and found that ω-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, ω-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403 → Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of ω-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the ω-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases.
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December 1998
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Research Article|
December 01 1998
ω-Oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site
Igor IVANOV;
Igor IVANOV
*Instiutute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Str. 3-4, D-10115 Berlin, Germany
†M. V. Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, 117571 Moscow, Russia
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Kristin SCHWARZ;
Kristin SCHWARZ
*Instiutute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Str. 3-4, D-10115 Berlin, Germany
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Herman G. HOLZHÜTTER;
Herman G. HOLZHÜTTER
*Instiutute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Str. 3-4, D-10115 Berlin, Germany
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Galina MYAGKOVA;
Galina MYAGKOVA
†M. V. Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, 117571 Moscow, Russia
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Hartmut KÜHN
Hartmut KÜHN
1
*Instiutute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Str. 3-4, D-10115 Berlin, Germany
1To whom correspondence should be addressed (e-mail hartmut.kuehn@rz.hu-berlin.de).
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Biochem J (1998) 336 (2): 345–352.
Article history
Received:
June 15 1998
Revision Received:
August 10 1998
Accepted:
September 18 1998
Citation
Igor IVANOV, Kristin SCHWARZ, Herman G. HOLZHÜTTER, Galina MYAGKOVA, Hartmut KÜHN; ω-Oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site. Biochem J 1 December 1998; 336 (2): 345–352. doi: https://doi.org/10.1042/bj3360345
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