Although the human O6-alkylguanine-DNA alkyltransferase (AGT) is very sensitive 
to inactivation by O6-benzylguanine (BG) or 
2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (5-nitroso-BP), the equivalent 
protein formed by the carboxyl terminal domain of the product of the Escherichia 
coli ada gene (Ada-C) is unaffected by these inhibitors. This difference is 
remarkable in view of the substantial similarity between these proteins (33% of 
the residues in the common sequence are identical) and is potentially very 
important since these inhibitors are under development as drugs to enhance the 
anti-tumor activity of alkylating agents. In order to understand the reason for 
the resistance of the Ada-C protein, we have made chimeras between Ada-C and AGT 
sequences and mutations in the Ada-C protein, expressed the altered proteins in 
an E. coli strain lacking endogenous alkyltransferase activity and tested the 
inactivation of the resulting proteins by BG or 5-nitroso-BP. Chimeric 
alkyltransferase proteins were made in which the residues on the amino side of 
the cysteine acceptor site came from Ada-C and the residues on the carboxyl side 
came from AGT and vice versa but these did not show sensitivity to BG suggesting 
that resistance is produced by residues in both segments of the protein. 
Analysis of the Ada-C mutant proteins revealed two sites for mutations that 
confer sensitivity to these inhibitors. One of these was tryptophan-336 and the 
other was residues lysine-314 and alanine-316. Thus, when the combined mutations 
of A316P/W336A were made in the Ada-C sequence, the protein was sensitive to 
inactivation by BG. This A316P/W336A mutant protein was even more sensitive to 
5-nitroso-BP and the mutant proteins W336A, K314P/A316P and A316P could also be 
inhibited by this drug (in decreasing order of sensitivity) although the control 
Ada-C and a mutant R335S were not inhibited. These results provide strong 
support for the hypothesis that the resistance of the Ada-C alkyl-transferase is 
due to a steric effect limiting access to the active site. Insertion of proline 
residues at positions 314 and 316 and removal of the bulky tryptophan residue at 
position 336 increases the space available at the active site and permits these 
inhibitors to be effective.