Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V.
Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the alpha-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.
Manuel
R C
RC
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555.
Latham
K A
KA
Dodson
M L
ML
Lloyd
R S
RS
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004274:DNA, Recombinant; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011740:Pyrimidine Dimers / Q000378:metabolism; D013941:Thymine / Q000378:metabolism; D014764:Viral Proteins
D004274:DNA, Recombinant; D011740:Pyrimidine Dimers; D014764:Viral Proteins; D018698:Glutamic Acid; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D013941:Thymine