O6-Alkylguanine-DNA alkyltransferase repairs damaged DNA by transferring an alkyl chain from the O6-position of guanine to a cysteine on the protein. This action produces the original DNA but the enzyme's free cysteine is not regenerated and the enzyme activity is destroyed. The formation and persistence of the O6-methylguanine lesion has been implicated in carcinogenesis. The mechanism by which the protein effects methyl transfer is not known. The postulated mechanism of action of AGT which will be tested in this proposal is as follows. AGT activates the cysteine as a nucleophile by deprotonation. The guanine is enhanced as a leaving group by protonation of a heteroatom. The cysteine attacks the methyl group displacing the guanine in a concerted reaction. The direct transfer of the methyl group from the DNA to the cysteine will be investigated by determining whether all three hydrogens are transferred with the methyl group and by determining the stereochemistry of the methyl transfer. The activation of guanine as a leaving group by protonation of a nitrogen on the nucleobase will be probed by synthesizing and then reacting O6-methyldeazaguanine substrate analogs with AGT. Low reactivity of a particular O6-methyldeazaguanine substrate will implicate that nitrogen as a proton acceptor. To determine if AGT activates guanine as a leaving group by protonation of the oxygen at the 6-position on the nucleoside, S6-methyl-6-thioguanine and Se6-methyl-6-selenoguanine substrate analogs will be synthesized and reacted with AGT. Analysis of the kinetic parameters will indicate whether the protein protonates the oxygen at the 6-position. The protonation state of the cysteine residue will be probed by investigating the pH profile of reactivity of AGT with the substrates and with iodoacetamide. The identity of possible basic amino acids in the active site of AGT which could deprotonate the cysteine will be investigated by synthesizing and then reacting AGT with active site-directed alkylating agents.
