Fe(III)-lipoxygenase converts its suicide-type inhibitor octadeca-9,12-diynoic acid into 11-oxooctadeca-9,12-diynoic acid.
Triple bond analogues of polyunsaturated fatty acids irreversibly inactivate lipoxygenases. During the inactivation the inhibitors are converted enzymatically [Kühn, H., et al. (1984) Eur. J. Biochem. 139, 577-583]. Since the converted inhibitor molecules may hold important information about the inactivation mechanism, we have determined the structure of the product that is formed during the irreversible inactivation of soybean lipoxygenase-1 by octadeca-9,12-diynoic acid (ODYA), the triple bond analogue of linoleic acid. This product is formed only in the presence of Fe(III)-lipoxygenase-1 and O2. It was purified by C18 solid phase extraction and reversed phase HPLC and was identified with UV, IR, and NMR spectroscopic and mass spectrometric techniques as the novel lipoxygenase product, 11-oxooctadeca-9,12-diynoic acid (11-oxo-ODYA). It is estimated that each lipoxygenase molecule produces 8-10 11-oxo-ODYA molecules before it is inactivated. Furthermore, we have shown that in a secondary reaction 3-4 molecules of 11-oxo-ODYA are covalently attached per lipoxygenase molecule, most likely, to solvent-exposed amino groups. This leads to the formation of a N-penten-4-yn-3-one chromophore, RC(NHX)=CHC(O)C=CR1, in which X stands for the protein and R or R1 for CH3(CH2)4- or -(CH2)7COOH, respectively. Fe(II)- and Fe(III)-lipoxygenase remain active upon reaction with purified 11-oxo-ODYA. It is concluded that (a) several enzymatic turnovers are required for the complete inactivation of lipoxygenase by ODYA and (b) covalent attachment of 11-oxo-ODYA occurs outside the active site and is not the cause of the inactivation.
Nieuwenhuizen
W F
WF
Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Schilstra
M J
MJ
van der Kerk-Van Hoof
A
A
Brandsma
L
L
Veldink
G A
GA
Vliegenthart
J F
JF
D000480:Alkynes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D053280:Diynes; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005290:Ferric Compounds / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013025:Glycine max / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
C095414:11-oxooctadeca-9,12-diynoic acid; D000480:Alkynes; D053280:Diynes; D005231:Fatty Acids, Unsaturated; D005290:Ferric Compounds; D016859:Lipoxygenase Inhibitors; C095413:octadeca-9,12-diynoic acid; D008084:Lipoxygenase