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

Research output: Contribution to journalArticle

55 Citations (Scopus)

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
StatePublished - Sep 23 2003

Fingerprint

Papain
Cysteine
Dinitrochlorobenzene
Biochemistry
Cross Reactions
DNA Repair
Anions
Glutathione
Catalyst activity
Catalytic Domain
Repair
DNA
Substrates
DNA alkyltransferase
4,4'-dipyridyl disulfide

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Guengerich, F. Peter ; Fang, Qingming ; Liu, Liping ; Hachey, David L. ; Pegg, Anthony E. / O6-alkylguanine-DNA alkyltransferase : Low pKa and high reactivity of cysteine 145. In: Biochemistry. 2003 ; Vol. 42, No. 37. pp. 10965-10970.
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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.",
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O6-alkylguanine-DNA alkyltransferase : Low pKa and high reactivity of cysteine 145. / Guengerich, F. Peter; Fang, Qingming; Liu, Liping; Hachey, David L.; Pegg, Anthony E.

In: Biochemistry, Vol. 42, No. 37, 23.09.2003, p. 10965-10970.

Research output: Contribution to journalArticle

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AU - Guengerich, F. Peter

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AU - Liu, Liping

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AU - Pegg, Anthony E.

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