MECHANISM OF INACTIVATION OF HUMAN O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE BY O6-BENZYLGUANINE

Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransfe rases from the E. coli ada gene or from yeast were not affected. O6-Be nzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltr ansferases at 200 muM in 30 min, slightly affected the Ada protein, an d had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivati on of the human alkyltransferase by O6-benzylguanine led to the format ion of S-benzylcysteine in the protein and to the stoichiometric produ ction of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6- benzylguanine inactivates the protein by acting as a substrate for alk yl transfer and by forming S-benzylcysteine at the acceptor site of th e protein. The inability of O6-benzylguanine to inactivate the microbi al alkyltransferases may be explained by steric constraints at this si te. The reduced effectiveness of the allyl compared to the benzyl deri vative is in accord with its expected, lower rate of participation in bimolecular displacement reactions, and its ability to inactivate the Ogt alkyltransferase may be explained by its smaller size permitting a ccess to this active site. These studies unequivocally show that the h uman alkyltransferase protein can act on low molecular weight substrat es lacking the polynucleotide structure. In addition to their use to i nactivate the protein, such substrates may also prove useful for the a ssay of mammalian alkyltransferase.