Site-directed mutagenesis of glutamic acid 172 to glutamine completely inactivated human O6-alkylguanine-DNA-alkyltransferase.

DNA repair by O6-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O6-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O6-alkylguanine-DNA-alkyltransferase (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase activity of the protein. This suggests that glutamic acid 172 is crucial to the alkyl transfer. It may act as a general acid (as CO2H) or base (as CO2-), or have a role as a component of a salt-link (-CO2-.....+N-), vital for the structural integrity of the active site. This is the first mutational inactivation of a protein in this family of DNA repair molecules by means of a residue change outside the highly conserved pentet (PCHRV) which includes the active-site cysteine.