Submitted on May 4, 2007
Revised on October 26, 2007
Accepted on October 26, 2007
Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal
Erin M. Shonsey, Shannon M. Eliuk, Michelle S. Johnson, Stephen Barnes, Charles N. Falany, Victor M. Darley-Usmar, and Matthew B. Renfrow
Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294
Corresponding Author: 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 Apo E knockout 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. Since 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 M) 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.
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