Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Publisher Website
Google Scholar
Abstract
Pyridoxal-5''-phosphate (PLP) is an inhibitor of DNA polymerase activity of E. coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP (deoxynucleotide triphosphate), and Hill plot analysis revealed that 2 molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with NaB3H4 resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. Thus, a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.