Amino acid residues affecting the activity and stability of human O6-alkylguanine-DNA alkyltransferase.

Amino acid residues in the human O6-alkylguanine-DNA alkyltransferase (AGT) were mutated and seventeen of the mutant proteins expressed in the ada- ogt-E. coli strain GWR 109 which is very sensitive to killing by methylating agents because of the absence of endogenous alkyltransferases. Thirteen of the mutations tested (delta-10, delta 1-19, R128A, N137A, H146A, R147A, delta N157, Y158A, E172Q, delta 92-97, Y114E, C145A and E172stop) reduced activity to below detectable levels when crude cell extracts were tested for the ability to remove O6-[3H]methylguanine from 3H-methylated DNA. However, only 4 of these mutations (delta 92-97, Y114E, C145A and E172stop) led to a complete loss of activity when tested for the ability to protect the cells from killing by MNNG. This suggests that the other nine mutations do not lead to the complete inactivation of AGT but produce protein with a reduced activity or in reduced amounts. These results show that none of the residues altered in these mutations (delta 1-10, delta 1-19, R128A, N137A, delta N157, H146A, R147A, Y158A and E172Q) are absolutely essential for AGT activity in protection against killing by MNNG. The stability of the mutant AGT proteins was determined by measuring the half-life of the protein synthesis was blocked. These results indicated that five of mutants that lacked AGT activity when tested in the crude extracts (Y114E, R128A, C145A, delta N157 and Y158A) were stable in the cell showing that the alteration of these residues does greatly reduce AGT activity. The other eight mutants lacking activity in crude extracts (delta 1-10, delta 92-97, E172Q, E172stop, delta 1-19, N137A, H146A and R147A) produced a large decrease in the stability of the AGT protein. This may account for the inability to detect AGT activity in vitro despite the ability to protect from MNNG toxicity in vivo. It is of particular interest that mutation of residues His146, Arg147, Asn137 and Glu172 resulted in unstable AGT proteins active in vivo but not in vitro. The crystal structure of the related Ada-C alkyltransferase suggests the involvement of these residues with the Cys145 acceptor site in a hydrogen bond network that may stabilize the protein and aid in the reaction mechanism. The data presented here support the existence of such an interaction existing in the human AGT and stress its importance in maintaining the configuration of the protein.