THE ROLE OF TYROSINE-158 IN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY

The tyrosine residue present at position 158 in the human O-6-alkylgua nine-DNA alkyltransferase is one of 22 amino acid residues that are co nserved in all known alkyltransferase protein sequences, The importanc e of this amino acid in the reactions brought about by the alkyltransf erase was studied by changing this residue to alanine or to phenylalan ine. The control and mutant alkyltransferase proteins were expressed i n an Escherichia coli strain lacking alkyltransferase activity and the proteins purified to near homogeneity and their activities measured u sing both methylated DNA and O-6-benzylguanine (BG) as substrates. The alteration to alanine led to a very large decrease in activity toward s both substrates but removal of O-6-methylguanine from DNA and the co nversion of BG to guanine could still be detected when large amounts o f 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 met hylated DNA only slightly (to about one-third). The conversion of BG t o 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 o f DNA. These results suggest that the presence of tyrosine at position 158 plays an important but not absolutely essential role in the react ions brought about by the alkyltransferase. This role is likely to inv olve the stabilization of the bound substrate by interaction with the aromatic ring of the tyrosine. The hydrogen bond formed by the hydroxy l group from tyrosine-158 may also facilitate the reaction but the con tribution from this interaction is relatively small.