The role of the conserved residue Glu-57 in the mechanism of the MutT enzyme from Escherichia coli was investigated by mutagenesis and heteronuclear NMR methods. The enzymatic activity of the E57Q mutant is at least 10(5)-fold lower than that of the wild type enzyme. The solution structure of the E57Q mutant, based on comparisons of 1H-15N NOESY HSQC spectra and 1H-15N HSQC spectra to those of the wild type enzyme, differs in a region near Glu-57. The dissociation constants (KD) of the E-Mg2+ and E-Mn2+ complexes increased 3.3- and 3.6-fold, respectively, in the E57Q mutant, while the KD of E-dGTP is unaltered from that of the wild type enzyme. The enhanced paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons is halved in the E57Q mutant indicating an altered metal-binding site. 1H-15N HSQC titrations of E57Q with MnCl2 show selective attenuation of the side chain NH signals of Gln-57 and the backbone NH signals of Gly-37, Gly-38, Lys-39, Glu-53, Glu-56, Gln-57, and Glu-98, indicating proximity of bound Nm2+ to these residues. The same resonances of the wild type and the E57Q mutant enzymes are attenuated by Mn2+, but significantly smaller paramagnetic effects (relative to the largest effect on Lys-39) are found on Gly-37, Gly-38, Val-58, and Glu-98 of the mutant, indicating an altered position of the bound divalent cation. Thus Glu-57 is probably a ligand to the enzyme-bound metal, and the profound loss of catalytic activity in the E57Q mutant results from structural and electronic changes at the site of the enzyme-bound divalent cation. 1H-15N HSQC titrations of the wild type enzyme with MgCl2 show changes in chemical shifts of 15N and NH resonances in regions closely overlapping those induced by the E57Q mutation itself, suggesting that the loss of the negative charge at Glu-57, either by mutation or by neutralization with Mg2+, induces a similar effect. In the E57Q mutant, the slow exchange of the side chain NH2 protons of Gln-57 and NOE's from the NH2 protons of Gln-57 to the beta and gamma protons of Glu-98 suggests hydrogen bonding of Gln-57 to Glu-98 in the free enzyme. 1H-15N HSQC titrations of both the wild type and mutant enzymes with dGTP show changes in 15N and NH chemicals shifts of residues in a cleft formed by beta-strands A, C, and D on one side and loop I, the end of loop IV, and the beginning of helix II on the other side, suggesting this cleft to be the nucleotide binding site. These changes in chemical shift were smaller or absent in titrations of the wild type or mutant enzymes with AMPCPP or Mg2+-AMPCPP, in accord with the strong preference of the MutT enzyme for guanine over adenine nucleotide substrates.