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 ye t biochemically separable functions, Previous studies using chemical m odification and site-directed mutagenesis techniques have shown that t he catalytic activities are mediated through the alpha-amino group of the enzyme forming a covalent (imino) intermediate, However, in additi on to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 ne ar the active site, and this residue has been strongly implicated in t he reaction chemistry, In order to understand the role of Glu-23 in th e reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D ) were constructed, and the mutant proteins were evaluated for DNA gly cosylase 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, whil e replacement with Asp retained negligible amounts of glycosylase acti vity, but retained near wild type levels of AP lyase activity, Gel shi ft assays revealed that all four mutant proteins can recognize and bin d to thymine dimers, The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely t o require an acidic group in the active site of the enzyme.