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

F. Peter Guengerich, Qingming Fang, Liping Liu, David L. Hachey, Anthony E. Pegg

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Abstract

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 pKa was estimated from plots of UV spectra (A 239) and reactivity toward 4,4'-dithiopyridine vs pH. The estimated pKa 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 pKa 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.

Original languageEnglish (US)
Pages (from-to)10965-10970
Number of pages6
JournalBiochemistry
Volume42
Issue number37
DOIs
Publication statusPublished - Sep 23 2003

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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