A carboxylate triad is essential for the polymerase activity of Escherichia coli DNA polymerase I (Klenow fragment) - Presence of two functional triads at the catalytic center

The catalytic roles of two essential active-site aspartates at positions 70 5 and 882 of Escherichia coli DNA polymerase I have been well established ( Steitz, T. A. (1998) Nature 391, 231-232), We now demonstrate that the part icipation of at least one additional carboxylate, a glutamate at position 7 10 or 883, is obligatory for catalysis. This conclusion has been drawn from our investigation of the properties of single (E710D, E710A, E883D, and E8 83A) and double (E710D/E883D and E710A/E883A) substitutions of residues Glu (710) and Glu(883). While single substitutions of either of the glutamates resulted in some reduction in polymerase activity, the mutant enzyme with s imultaneous substitution of both glutamates with alanine exhibited a nearly complete loss of activity. Interestingly, substitution with two aspartates in place of the glutamates resulted in an enzyme species that catalyzed DN A synthesis in a strictly distributive mode. Pyrophosphorolytic activity of the mutant enzymes reflected their polymerase activity profiles, with mark edly reduced pyrophosphorolysis by the double mutant enzymes. Moreover, an evaluation of Mg2+ and salt optima for all mutant enzymes of Glu(710) and G lu(883) revealed significant deviations from that for the wild type, implyi ng a possible role of these glutamates in metal coordination as well as in maintaining the structural integrity of the active site.