Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I
- 1 March 1987
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 26 (6) , 1704-1709
- https://doi.org/10.1021/bi00380a032
Abstract
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 in 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.This publication has 12 references indexed in Scilit:
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