Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase

The anticancer drug caracemide, N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1. No effect on the smaller protein R2 was observed. The effect of the degradation product was about 30 times lower than that of caracemide itself. The caracemide inactivation of R1 is irreversible, with an apparent second-order rate constant of 150 M-1 s-1. The R1R2 holoenzyme was approximately 30 times more sensitive to caracemide inactivation than the isolated R1 protein. The ribonucleotide reductase substrates were potent competitors of the caracemide inhibition, with a K(diss) for GDP binding to R1 of 80-mu-M. The reducing agent dithiothreitol was also found to be a potent competitor of caracemide inactivation. These results indicate that caracemide inactivates R1 by covalent modification at the substrate-binding site. By analogy with the known interaction between caracemide and acetylcholinesterase or choline acetyltransferase, we propose that the modification of R1 occurs at an activated cysteine or serine residue in the active site of the enzyme.