Reaction of O⁶-Alkylguanine-DNA Alkyltransferase with O⁶-Methylguanine Analogues: Evidence That the Oxygen of O⁶-Methylguanine is Protonated by the Protein To Effect Methyl Transfer

The DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs the promutagenic O⁶methylguanine lesion by transferring the methyl group to a cysteine residue on the protein. A mechanism in which AGT activates the guanyl moiety as a leaving group by protonation of a heteroatom on guanine was probed by reacting AGT with analogues of (Amethylguanine in which the heteroatoms were changed. The initial rates of reaction were measured at various substrate concentrations in 50 mM Hepes, 1 mM EDTA, 1 mM DTT, and 10 % glycerol, pH 7.8 at 37 °C. The k_inact h^-1) and K_in (mM) were determined for O⁶-methylguanine (1.66 ± 0.19, 1.51 ± 0.32), 6-methoxypurine (1.07 ± 0.25, 10.6 ± 4.2), S⁶-methyl-6-thioguanine (0.63 ± 0.04, 1.17 ± 0.18), 6-methylthiopurine (no reaction), Se⁶-methyl-6-selenoguanine (1.76 ± 0.28, 10.6 ± 5.0), 6-methylselenopurine (2.51 ± 0.62, 15.7 ± 6.3), (O⁶-methyl-1-deazaguanine (1.71 ± 0.34, 14.8 ± 4.4), O⁶-methyl-3-deazaguanine (1.90 ± 0.24, 2.54 ± 0.59), and O⁶-methyl-7-deazaguanine (1.97 ± 0.26, 2.56 ± 0.72). These results indicate that replacement of the nitrogens does not affect the K_inact parameter but the K_in is increased upon removal of the exocyclic amino group and the nitrogen at the 1-position. Replacement of the oxygen with sulfur decreases the K_inact, and replacement with selenium increases the K_in. The results are consistent with a mechanism in which O⁶-methylguanine binds to the active site of AGT with hydrogen bonds to the oxygen, the exocyclic amino group, and the nitrogen at the 1-position of the substrate. The methyl group is then displaced from the guanine as a proton is transferred to the oxygen, neutralizing the charge on the leaving group.