Mutational and pH studies of the 3 '-> 5 ' exonuclease activity of bacteriophage T4 DNA polymerase

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B, Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase ind icate that residue Asp-324, which binds metal ion A, is the single most imp ortant residue for the hydrolysis reaction. In the absence of a nonenzymati c source of hydroxide ions, an alanine substitution for residue Asp-324 red uced exonuclease activity 10-100-fold more than alanine substitutions for t he other metal-binding residues, Asp-112 and Asp-219, Thus, exonuclease act ivity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are c onsistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiest er bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist th e reaction by lowering the pK(a) of the metal ion-A coordinated water molec ule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.