A unique conformation of deoxynucleoside triphosphate substrates bound to Escherichia coli DNA polymerase I has been determined by nuclear magnetic resonance techniques. The effects of Mn(II) bound at the active site of the enzyme on the longitudinal (T1p-1) and transverse (T2p-1) relaxation rates of the alpha, beta, and gamma phosphorus atoms and 5 protons of enzyme-bound thymidine 5'-triphosphate (dTTP) were measured at 40.5 MHz (31P), 100 and 220 MHz (1H). From frequency dependence of T1p-1, a correlation time of 7 X 10(-10) s and Mn(II) to proton distances of 10.4, 9.9, 10.3, 10.8, and 8.4 A were calculated for the --CH3, H6, H'1, H'2, and H'4 protons. The calculated Mn(II) to phosphorus distances of 4.2, 4.8, and 3.2 A for the alpha, beta, and gamma phosphorus atoms indicates that Mn(II) corrdinates directly only with the gamma-phosphoryl group and that a puckered triphpsphate conformation exists for the enzyme-bound dTTP. This differs from the binary Mn(II)-dTTP complex in which alpha, beta, and gamma phosphoryl coordination occurs, and a thymine-deoxyribose torsion angly (chi) about the glycosidic bond of 40 degrees is detected. The eight manganese-substrate distances on the enzyme are fit by a unique Mn-dTTP conformation, with a torsion angle equal to 90 degrees, indistinguishable from that found for a deoxynucleotidyl unit in double helical DNA-B. Hence, binding to DNA polymerase appears to adjust the conformation of dTTP for Watson-Crick basepairing. Similarly, the binding of Mn-dATP to DNA polymerase I increased the distances from Mn(II) to the H2, H8, H'1, and H'4 protons of dATP but the adenine-deoxyribose torsion angle of 90 degrees was preserved. Such preorientation of substrates could facilitate incorporation of the complementary nucleotide. When positioned within the DNA structure, the conformation of enzyme-bound Mn-dTTP requires an inline nucleophilic attack on the alpha phosphorus with Mn(II) promoting pyrophosphate departure.