Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V

摘要: Abstract 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 site-directed mutagenesis techniques have shown that the catalytic activities mediated through α-amino group of enzyme forming a covalent (imino) intermediate. However, in addition to amino-terminal active site residue, examination x-ray crystal structure reveals presence Glu-23 near site, this residue been strongly implicated reaction chemistry. In order understand role mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, mutant proteins evaluated for AP defined substrates specific vitro vivo assays. Replacement with Gln, Cys, or His completely abolished while replacement Asp retained negligible amounts activity, but wild type levels activity. Gel shift assays revealed all can recognize bind thymine dimers. The results indicate is candidate stabilizing charge imino intermediate likely require an acidic enzyme.