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Journal of Lipid Research, Vol. 49, 282-294, February 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

E. M. Shonsey^1,, S. M. Eliuk, M. S. Johnson, S. Barnes^*,,**,, C. N. Falany, V. M. Darley-Usmar^, and M. B. Renfrow^*,

^* Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294
Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294
^** Comprehensive Cancer Center Mass Spectrometry Shared Facility, University of Alabama at Birmingham, Birmingham, AL 35294
Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294
University of Alabama Biomedical Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry Laboratory, University of Alabama at Birmingham, Birmingham, AL 35294

Published, JLR Papers in Press, October 27, 2007.

¹ To whom correspondence should be addressed. e-mail: eshonsey{at}uab.edu

The hepatic enzyme bile acid CoA:amino acid N-acyltransferase (BAT) catalyzes the formation of amino acid-conjugated bile acids. In the present study, protein carbonylation of BAT, consistent with modification by reactive oxygen species and their products, was increased in hepatic homogenates of apolipoprotein E knock-out mice. 4-Hydroxynonenal (4HNE), an electrophilic lipid generated by oxidation of polyunsaturated long-chain fatty acids, typically reacts with the amino acids Cys, His, Lys, and Arg to form adducts, some of which (Michael adducts) preserve the aldehyde (i.e., carbonyl) moiety. Because two of these amino acids (Cys and His) are members of the catalytic triad of human BAT, it was proposed that 4HNE would cause inactivation of this enzyme. As expected, human BAT (1.6 µM) was inactivated by 4HNE in a dose-dependent manner. To establish the sites of 4HNE's reaction with BAT, peptides from proteolysis of 4HNE-treated, recombinant human BAT were analyzed by peptide mass fingerprinting and by electrospray ionization-tandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dose-dependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.

Supplementary key words coenzyme A • post-translational modifications • mass spectrometry

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