10.1021/tx700271j.s002 Richard Coulter Richard Coulter Maureen Blandino Maureen Blandino Jessica M. Tomlinson Jessica M. Tomlinson Gary T. Pauly Gary T. Pauly Magdalena Krajewska Magdalena Krajewska Robert C. Moschel Robert C. Moschel Lisa A. Peterson Lisa A. Peterson Anthony E. Pegg Anthony E. Pegg Thomas E. Spratt Thomas E. Spratt Differences in the Rate of Repair of <i>O</i><sup>6</sup>-Alkylguanines in Different Sequence Contexts by <i>O</i><sup>6</sup>-Alkylguanine-DNA Alkyltransferase American Chemical Society 2007 AGT sequence contexts kinact sequence context alkyl groups Different Sequence Contexts 2007-12-17 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Differences_in_the_Rate_of_Repair_of_i_O_i_sup_6_sup_Alkylguanines_in_Different_Sequence_Contexts_by_i_O_i_sup_6_sup_Alkylguanine_DNA_Alkyltransferase/2968678 <i>O</i><sup>6</sup>-Alkylguanine-DNA alkyltransferase (AGT) repairs <i>O</i><sup>6</sup>-alkylguanine residues at different rates depending on the identity of the alkyl group as well as the sequence context. To elucidate the mechanism(s) underlying the differences in rates, we examined the repair of five alkyl groups in three different sequence contexts. The <i>k</i><sub>inact</sub> and <i>K</i><sub>m</sub> values were determined by measuring the rates of repair of oligodeoxynucleotide duplexes containing the <i>O</i><sup>6</sup>-alkylguanine residues with various concentrations of AGT in excess. The time course of the reactions all followed pseudo-first-order kinetics except for one of the <i>O</i><sup>6</sup>-ethylguanine substrates, which could be analyzed in a two-phase exponential equation. The differences in rates of repair between the different alkyl groups and the different sequence contexts are dependent on rates of alkyl transfer and not substrate recognition. The relative rates of reaction are in general benzyl > methyl > ethyl > 2-hydroxyethyl > 4-(3-pyridyl)-4-oxobutyl, but the absolute rates are dependent on sequence. The <i>k</i><sub>inact</sub> values between benzyl and 4-(3-pyridyl)-4-oxobutyl range from 2300 to 350000 depending on sequence. The sequence-dependent variation in <i>k</i><sub>inact</sub> varied the most for <i>O</i><sup>6</sup>-[4-(3-pyridyl)-4-oxobutyl]guanine, which ranged from 0.022 to 0.000016 s<sup>−1</sup>. The results are consistent with a mechanism in which the <i>O</i><sup>6</sup>-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context.