O⁶-alkylguanine-DNA alkyltransferase: Low pK_a and high reactivity of cysteine 145

The active site cysteine of human O⁶-alkylguanine-DNA alkyltransferase (hAGT), Cys145, was shown to be highly reactive with model electrophiles unrelated to substrates, including 1-chloro-2,4-dinitrobenzene. The high reactivity suggested that the Cys145 thiolate anion might be stable at neutral pH. The pK_a was estimated from plots of UV spectra (A ₂₃₉) and reactivity toward 4,4'-dithiopyridine vs pH. The estimated pK_a for hAGT was 4-5, depending upon the method used, and near that of the extensively characterized papain Cys25. Rates of reaction with 4,4'-dithiopyridine were similar for the thiolate forms of hAGT, papain, glutathione, and the bacterial hAGT homologue Ogt (the pK_a of the latter was 5.4). Bound Zn²⁺ has previously been shown to be required for the catalytic activity of hAGT (Rasimas, J. J. et al. (2003) Biochemistry 42, 980-990). Zn²⁺ was shown to be required for the low pK _a of hAGT. The high reactivity of hAGT Cys145 is postulated to be important in normal catalytic function, in cross-linking reactions involving bis-electrophiles, and in inhibition of the DNA repair function of hAGT by electrophiles.