Gas-Phase Studies of Purine 3-Methyladenine DNA Glycosylase II (AlkA) Substrates

Abstract

3-Methyladenine DNA glycosylase II (AlkA) is an enzyme that cleaves a wide range of damaged bases from DNA. The gas-phase thermochemical properties (tautomerism, acidity, and proton affinity) have been measured and calculated for a series of AlkA purine substrates (7-methyladenine, 7-methylguanine, 3-methyladenine, 3-methylguanine, purine, 6-chloropurine, xanthine) that have not been heretofore examined. The damaged nucleobases are found to be more acidic than the normal nucleobases adenine and guanine. Because of this increased acidity, the damaged bases would be expected to be more easily cleaved from DNA by AlkA (their conjugate bases should be better leaving groups). We find that the gas-phase acidity correlates to the AlkA excision rates, which lends support to an AlkA mechanism wherein the enzyme provides a nonspecific active site, and nucleobase cleavage is dependent on the intrinsic N-glycosidic bond stability.

Gas‐Phase Studies of Formamidopyrimidine Glycosylase (Fpg) Substrates

“…The calculateda cidity and protona ffinity values for OHU (6) and DHT (7)a re summarized in Ta ble5 (DH values listed;f or DG values, see the Supporting Information). For acidity,the calculated and experimental values are quite similar; for OHU, the experimental value is 2.6 kcal mol À1 highert han the calculated, whereas for DHT,t he experimental value is 1.9 kcal mol À1 higher.…”

“…The inability to accurately calculate PAs, even with more intensive computational methods such as M062X and CBS, has been observed by our group before in the study of two other nucleobases,u racil and xanthine. [6,23] All nucleobase structures that show this discrepancy betweencalculated and experimentalP As have astructural element in common: the site being protonated has an NH group b to the protonation site and ad ouble bond in the ring. DHT (7)d oes not have the double bond at C4ÀC5.…”

Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA

“…58 Notably, it was also proposed that AAG excises some lesions without acid catalysis, such that the base departs as an anion; the glycosidic nitrogen (N9) is more acidic for Hx and εA relative to A and G in the gas phase, and this trend may hold in a hydrophobic active site, which could make Hx and εA more susceptible to enzymatic excision. 56, 58, 59 …”

“…The glycosidic nitrogen of Hx is more acidic (p K a N9 8.9) than that of U (p K a N1 9.8), suggesting that the Hx anion could be a good leaving group. 39, 56 The glycosidic nitrogen of X is even more acidic (p K a N9 7.3, calculated), thus the X monoanion is predicted to be an excellent leaving group (Fig. 7).…”

“…7). 56, 79 However, N3-H is also relatively acidic, with a p K a N3 of 5.7 for the xanthosine nucleoside, 80 such that 2’-deoxyxanthosine (dX) is predominantly anionic at pH 7.4 (Fig. 7).…”