Divalent cation-dependent pyridoxal 5'-phosphate inhibition of Rauscher leukemia virus DNA polymerase: characterization and mechanism of action.
We have shown that pyridoxal 5'-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn2+, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.
D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis; D003854:Deoxyribonucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D011925:Rauscher Virus / Q000201:enzymology*; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D008345:Manganese; D011732:Pyridoxal Phosphate; D004247:DNA; D008274:Magnesium