The role of tyrosine-158 in O6-alkylguanine-DNA alkyltransferase activity.
The tyrosine residue present at position 158 in the human O6-alkylguanine-DNA alkyltransferase is one of 22 amino acid residues that are conserved in all known alkyltransferase protein sequences. The importance of this amino acid in the reactions brought about by the alkyltransferase was studied by changing this residue to alanine or to phenylalanine. The control and mutant alkyltransferase proteins were expressed in an Escherichia coli strain lacking alkyltransferase activity and the proteins purified to near homogeneity and their activities measured using both methylated DNA and O6-benzylguanine (BG) as substrates. The alteration to alanine led to a very large decrease in activity towards both substrates but removal of O6-methylguanine from DNA and the conversion of BG to guanine could still be detected when large amounts of the protein were used. The activity of the Y158A mutant was at least 800 times less than that of the control alkyltransferase. The change of tyrosine-158 to phenylalanine reduced the rate of reaction with methylated DNA only slightly (to about one-third). The conversion of BG to guanine by the Y158F mutant was also reduced to about one-third when assayed in the absence of DNA and by about one-half in the presence of DNA. These results suggest that the presence of tyrosine at position 158 plays an important but not absolutely essential role in the reactions brought about by the alkyltransferase. This role is likely to involve the stabilization of the bound substrate by interaction with the aromatic ring of the tyrosine. The hydrogen bond formed by the hydroxyl group from tyrosine-158 may also facilitate the reaction but the contribution from this interaction is relatively small.
Edara
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S
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Goodtzova
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K
Pegg
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AE
D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010649:Phenylalanine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism*
D014316:Tritium; D014443:Tyrosine; D010649:Phenylalanine; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000409:Alanine