Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine

Anthony E. Pegg, Timothy L. Byers, Kristin Swenn, Michael Boosalis, Leona Samson, Robert C. Moschel, M. Eileen Dolan

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Abstract

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 µM 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. The reduced effectiveness of the allyl compared to the benzyl derivative is in accord with its expected, lower rate of participation in bimolecular displacement reactions, and its ability to inactivate the Ogt alkyltransferase may be explained by its smaller size permitting access to this active site. These studies unequivocally show that the human alkyltransferase protein can act on low molecular weight substrates lacking the polynucleotide structure. In addition to their use to inactivate the protein, such substrates may also prove useful for the assay of mammalian alkyltransferase.

Original languageEnglish (US)
Pages (from-to)11998-12006
Number of pages9
JournalBiochemistry
Volume32
Issue number45
DOIs
StatePublished - Jan 1 1993

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Alkyl and Aryl Transferases
Guanine
Proteins
Substrates
Yeast
Escherichia coli
O(6)-benzylguanine
DNA alkyltransferase
Genes
Yeasts
Polynucleotides
Deoxyguanosine

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Pegg, A. E., Byers, T. L., Swenn, K., Boosalis, M., Samson, L., Moschel, R. C., & Dolan, M. E. (1993). Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine. Biochemistry, 32(45), 11998-12006. https://doi.org/10.1021/bi00096a009
Pegg, Anthony E. ; Byers, Timothy L. ; Swenn, Kristin ; Boosalis, Michael ; Samson, Leona ; Moschel, Robert C. ; Dolan, M. Eileen. / Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine. In: Biochemistry. 1993 ; Vol. 32, No. 45. pp. 11998-12006.
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title = "Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine",
abstract = "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 µM 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. The reduced effectiveness of the allyl compared to the benzyl derivative is in accord with its expected, lower rate of participation in bimolecular displacement reactions, and its ability to inactivate the Ogt alkyltransferase may be explained by its smaller size permitting access to this active site. These studies unequivocally show that the human alkyltransferase protein can act on low molecular weight substrates lacking the polynucleotide structure. In addition to their use to inactivate the protein, such substrates may also prove useful for the assay of mammalian alkyltransferase.",
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Pegg, AE, Byers, TL, Swenn, K, Boosalis, M, Samson, L, Moschel, RC & Dolan, ME 1993, 'Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine', Biochemistry, vol. 32, no. 45, pp. 11998-12006. https://doi.org/10.1021/bi00096a009

Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine. / Pegg, Anthony E.; Byers, Timothy L.; Swenn, Kristin; Boosalis, Michael; Samson, Leona; Moschel, Robert C.; Dolan, M. Eileen.

In: Biochemistry, Vol. 32, No. 45, 01.01.1993, p. 11998-12006.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mechanism of Inactivation of Human O6-Alkylguanine-DNA Alkyltransferase by O6-Benzylguanine

AU - Pegg, Anthony E.

AU - Byers, Timothy L.

AU - Swenn, Kristin

AU - Boosalis, Michael

AU - Samson, Leona

AU - Moschel, Robert C.

AU - Dolan, M. Eileen

PY - 1993/1/1

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N2 - 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 µM 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. The reduced effectiveness of the allyl compared to the benzyl derivative is in accord with its expected, lower rate of participation in bimolecular displacement reactions, and its ability to inactivate the Ogt alkyltransferase may be explained by its smaller size permitting access to this active site. These studies unequivocally show that the human alkyltransferase protein can act on low molecular weight substrates lacking the polynucleotide structure. In addition to their use to inactivate the protein, such substrates may also prove useful for the assay of mammalian alkyltransferase.

AB - 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 µM 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. The reduced effectiveness of the allyl compared to the benzyl derivative is in accord with its expected, lower rate of participation in bimolecular displacement reactions, and its ability to inactivate the Ogt alkyltransferase may be explained by its smaller size permitting access to this active site. These studies unequivocally show that the human alkyltransferase protein can act on low molecular weight substrates lacking the polynucleotide structure. In addition to their use to inactivate the protein, such substrates may also prove useful for the assay of mammalian alkyltransferase.

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