REACTION OF O6-ALKYLGUANINE DNA ALKYLTRANSFERASE WITH O6-METHYLGUANINE ANALOGS - 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 O6-methylguanine 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 O6-methylguanine 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-degrees-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.