Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine.
Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)
Pegg
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AE
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Boosalis
M
M
Samson
L
L
Moschel
R C
RC
Byers
T L
TL
Swenn
K
K
Dolan
M E
ME
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; C002206:S-benzylcysteine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine