Nuclear Overhauser effect studies of the conformations and binding site environments of deoxynucleoside triphosphate substrates bound to DNA polymerase I and its large fragment.
The conformations and binding site environments of Mg2+TTP and Mg2+dATP bound to Escherichia coli DNA polymerase I and its large (Klenow) fragment have been investigated by proton NMR. The effect of the large fragment of Pol I on the NMR line widths of the protons of Mg2+TTP detected one binding site for this substrate with a dissociation constant of 300 +/- 100 microM and established simple competitive binding of deoxynucleoside triphosphates at this site in accord with previous equilibrium dialysis experiments with whole Pol I [Englund, P. T., Huberman, J.A., Jovin, T.M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038]. Primary negative nuclear Overhauser effects were used to calculate interproton distances on enzyme-bound Mg2+dATP and Mg2+TTP. These distances established that each substrate was bound with an anti-glycosidic torsional angle (chi) of 50 +/- 10 degrees for Mg2+dATP and 40 +/- 10 degrees for Mg2+TTP. The sugar pucker of both substrates was predominantly O1'-endo, with a C5'-C4'-C3'-O3' exocyclic torsional angle (delta) of 95 +/- 10 degrees for Mg2+dATP and 100 +/- 10 degrees for Mg2+TTP. The consistency of these conformations with those previously proposed, on the basis of distances from Mn2+ at the active site [Sloan, D. L., Loeb, L. A., Mildvan, A.S., & Feldman, R.J. (1975) J. Biol. Chem. 250, 8913], indicates a unique conformation for each bound nucleotide. The chi and delta values of the bound substrates are appropriate for nucleotide units of B DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
D001665:Binding Sites; D004256:DNA Polymerase I / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation
D003854:Deoxyribonucleotides; D010446:Peptide Fragments; D004256:DNA Polymerase I; D008274:Magnesium