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 705 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 participation of at least one additional carboxylate, a glutamate at position 710 or 883, is obligatory for catalysis. This conclusion has been drawn from our investigation of the properties of single (E710D, E710A, E883D, and E883A) 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 simultaneous 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 DNA synthesis in a strictly distributive mode. Pyrophosphorolytic activity of the mutant enzymes reflected their polymerase activity profiles, with markedly reduced pyrophosphorolysis by the double mutant enzymes. Moreover, an evaluation of Mg(2+) and salt optima for all mutant enzymes of Glu(710) and Glu(883) revealed significant deviations from that for the wild type, implying a possible role of these glutamates in metal coordination as well as in maintaining the structural integrity of the active site.
Gangurde
R
R
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
Kaushik
N
N
Singh
K
K
Modak
M J
MJ
D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010756:Phosphoric Acids / Q000378:metabolism; D010957:Plasmids; D012965:Sodium Chloride / Q000378:metabolism; D014162:Transfection; D014238:Trichloroacetic Acid / Q000378:metabolism
D010756:Phosphoric Acids; D001224:Aspartic Acid; D018698:Glutamic Acid; D012965:Sodium Chloride; D014238:Trichloroacetic Acid; D004247:DNA; D004256:DNA Polymerase I; D008274:Magnesium; D000409:Alanine