The inactivation of human and Escherichia coli O6-alkylguanine-DNA 
alkyltransferase by O6-methylguanine and O6-benzylguanine was compared. When 
HT29 cell extracts or E. coli Ada protein were incubated in the presence of 200 
microM O6-methylguanine for 1 h, alkyltransferase activity was reduced to 44 and 
39% of control levels respectively. However, under the same conditions 
O6-benzylguanine completely depleted alkyltransferase activity in the extract 
from human cells but had virtually no effect on the Ada protein. Incubation of 
the HT29 cell alkyltransferase with O6-benzyl[3H]guanine resulted in a 
time-dependent production of [3H]guanine. No similar production of [3H]guanine 
was observed in the presence of the Ada protein. In CHO cells transfected with 
the bacterial ada gene (CHO-ada) or the human alkyltransferase cDNA (CHO-MGMT), 
treatment with 500 microM O6-methylguanine inhibited both alkyltransferases by 
greater than 85%. In contrast, 2 microM O6-benzylguanine inhibited human 
alkyltransferase expressed in CHO-MGMT cells by greater than 99% though 
concentrations as high as 25 microM for 24 h had no inhibitory effects on the 
bacterial alkyltransferase expressed in CHO-ada cells. This selective inhibition 
was also observed in vivo in transgenic mice expressing ada in the liver where 
O6-benzylguanine caused a decrease of only 40% in total hepatic alkyltransferase 
activity compared to 95% in non-transgenic mice, consistent with inhibition of 
only the mammalian alkyltransferase and maintenance of bacterial 
alkyltransferase activity in these animals. Thus, while O6-methylguanine at high 
concentrations inactivates both bacterial and mammalian alkyltransferases, 
O6-benzylguanine is a substrate only for the mammalian protein and is unable, 
perhaps due to steric hindrance, to inhibit the Ada protein.