Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I.

Basu A, Modak MJ
Biochemistry (1987), Volume 26, Page 1704
PubMed entry


We have labeled the large fragment of Escherichia coli DNA polymerase ...
We have labeled the large fragment of Escherichia coli DNA polymerase I (Pol I) with pyridoxal 5'-phosphate, a substrate binding site directed reagent for DNA polymerases [Modak, M. J. (1976) Biochemistry 15, 3620-3626]. A covalent attachment of pyridoxal phosphate to Pol I results in the loss of substrate binding as well as the polymerase activity. The inactivation was found to be strictly dependent on the presence of a divalent metal ion. Four moles of pyridoxal phosphate was found to react per mole of the enzyme, while in the presence of substrate deoxynucleoside triphosphate only 3 mol of pyridoxal phosphate was bound. To identify the substrate-protected site on the enzyme, tryptic peptides from enzyme labeled with pyridoxal phosphate and tritiated borohydride, in the presence and absence of substrate, were resolved on a C-18 reverse-phase column. A single peptide containing the substrate-protected site was identified and further purified. The amino acid composition and sequence analysis of this peptide revealed it to span residues 756-775 in the primary acid sequence of Pol I. Lys-758 of this sequence was found to be the site of the pyridoxal phosphate reaction. It is therefore concluded that Lys-758 is the site of binding for the metal chelate form of nucleotide substrates in E. coli DNA polymerase I.




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