Citation

Nieuwenhuizen WF, Schilstra MJ, van der Kerk-Van Hoof A, Brandsma L, Veldink GA, Vliegenthart JF. Biochemistry 1995; 34(33): 10538-10545.

Copyright

(Copyright © 1995, American Chemical Society)

DOI

10.1021/bi00033a028

PMID

7654709

Abstract

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.

Language: en

Keywords

Alkynes; Chromatography, High Pressure Liquid; Diynes; Fatty Acids, Unsaturated; Ferric Compounds; Glycine max; Kinetics; Lipoxygenase; Lipoxygenase Inhibitors; Magnetic Resonance Spectroscopy; Mass Spectrometry; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared