Creatine kinase reversibly catalyzes the transfer of the high-energy phosphoryl group from phosphocreatine to MgADP for rapid regeneration of ATP. It is hypothesized that factors which perturb creatine kinase activity, such as reactive oxygen species resulting from oxidative stress, could have a major role in the pathogenesis of diseases, particularly in the brain, where the level of ATP utilization is high. The reactive aldehyde 4-hydroxy-2-nonenal is a major secondary product of lipid peroxidation caused by oxidative stress; the levels of both free and protein-bound 4-hydroxy-2-nonenal are increased in Alzheimer's disease brain. Preliminary reports indicated that creatine kinase had lower activity in Alzheimer's disease brain. In this study, we investigated the structural and functional consequences of reacting the cytosolic brain isoform of creatine kinase with 4-hydroxy-2-nonenal at pathophysiologically relevant concentrations of 4-hydroxy-2-nonenal (10-300 microM). Dose-dependent reduction of enzyme activity was observed and, for the first time, correlated with 4-hydroxy-2-nonenal adduct formation on specific amino acid residues, including the active site residues His66, His191, Cys283, and His296 as determined by Fourier transform-ion cyclotron resonance mass spectrometry.

Mass spectrometry with or without pre-analysis peptide fractionation can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen and electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighboring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4-hydroxynonenal (4HNE) of human bile acid CoA: amino acid N-acyltransferase (hBAT) and the cytosolic brain isoform of creatine kinase (CKBB) is associated with site-specific modifications. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) using nanoLC-electrospray ionization-mass spectrometry (ESI-MS) or direct infusion-ESI-MS with gas phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB, respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

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 mM) 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 ionizationtandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dosedependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.

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