Contribution of a conserved phenylalanine residue to the activity of Escherichia coli uracil DNA glycosylase.
Uracil DNA glycosylase (UDG) excises uracil from DNA to initiate repair of this lesion. This important DNA repair enzyme is conserved in viruses, bacteria, and eukaryotes. One residue that is conserved among all the members of the UDG family is a phenylalanine that stacks with uracil when it is flipped out of the DNA helix into the enzyme active site. To determine what contribution this conserved Phe residue makes to the activity of UDG, Phe-77 in the Escherichia coli enzyme was mutated to three different amino acid residues, alanine (UDG-F77A), asparagine (UDG-F77N), and tyrosine (UDG-F77Y). The effects of these mutations were measured on the steady-state and pre-steady-state kinetics of uracil excision in addition to enzyme.DNA binding kinetics. The overall excision activity of each of the mutants was reduced relative to the wild-type enzyme; however, each mutation gave rise to a different kinetic phenotype with different effects on substrate binding and catalysis. The excision activity of UDG-F77N was the most severely compromised, but this enzyme still bound to uracil-containing DNA at about the same rate as wild-type UDG. In contrast, the decrease in the excision activity of UDG-F77A is likely to reflect a greater reduction in uracil-DNA binding than in the catalytic step. Overall, the effects of the mutations on catalysis are best correlated with the polarity of the substituted residue such that an increase in polarity decreases the efficiency of uracil excision.
Shaw
Ryan W
RW
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610-0245, USA.
Feller
Joyce A
JA
Bloom
Linda B
LB
D002384:Catalysis; D017124:Conserved Sequence; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
D011994:Recombinant Proteins; D010649:Phenylalanine; D014498:Uracil; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase