O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145.
The active site cysteine of human O(6)-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 Zn(2+) has previously been shown to be required for the catalytic activity of hAGT (Rasimas, J. J. et al. (2003) Biochemistry 42, 980-990). Zn(2+) 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.
Guengerich
F Peter
FP
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. f.guengerich@vanderbilt.edu
Fang
Qingming
Q
Liu
Liping
L
Hachey
David L
DL
Pegg
Anthony E
AE
D000838:Anions; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D004260:DNA Repair; D004137:Dinitrochlorobenzene / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry*; D011725:Pyridines / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology
D000838:Anions; D004137:Dinitrochlorobenzene; D004220:Disulfides; D011725:Pyridines; C007542:4,4'-dipyridyl disulfide; D019853:O(6)-Methylguanine-DNA Methyltransferase; D015032:Zinc; D003545:Cysteine