Many enzymes in cells are modified covalently as a regulatory mechanism. For example, protein phosphorylation is a modification central to the regulation of metabolism and other cell functions. Another type of modification is ADP-ribosylation, best known from the modification of G-proteins by the bacterial enzymes cholera toxin and pertussis toxin, which are involved in the diseases cholera and whooping cough, respectively. Previous work has demonstrated the presence of cellular enzymes that catalyze reactions similar to those catalyzed by the bacterial toxins, and interest is now high in understanding how ADP-ribosylation catalyzed by endogenous enzymes may regulate animal cell function. The current work involves characterizing ADP-ribosylation of cysteine in cell-free systems. During the characterization of cysteine-specific ADP-ribosyltransferases, it was found that cysteine reacted with ADP-ribose nonenzymatically, to yield a product identified as ADP-ribosethiazolidine carboxylic acid. Protein cysteine residues were also ADP-ribosylated nonenzymatically, with a bond different from that formed with free cysteine, but similar to that formed by pertussis toxin-catalyzed ADP-riboyslation of G-proteins. Analysis of the chemical stability of ADP-ribosylcysteine in neutral hydroxylamine allows one to distinguish between enzymatic and nonenzymatic products. The identification of these nonenzymatic reactions is a key observation for the future investigation of this protein modification pathway in animal cells.
