title: Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction.
abstract: ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.
authors.affiliation: Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland 20892.
authors.forename: L J
authors.initials: LJ
authors.lastname: McDonald
authors.forename: L A
authors.initials: LA
authors.lastname: Wainschel
authors.forename: N J
authors.initials: NJ
authors.lastname: Oppenheimer
authors.forename: J
authors.initials: J
authors.lastname: Moss
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D037342:Pertussis Toxin; D051381:Rats; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D006898:Hydroxylamines; D013844:Thiazoles; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D019811:Hydroxylamine; D037342:Pertussis Toxin; D003545:Cysteine