Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using [U-13C]ATP and [U-13C]ADP.

A complete characterization of the conformations of Mn.ADP and Mn.ATP bound to the active site of yeast 3-P-glycerate kinase is presented. These conformations have been deduced on the basis of paramagnetic effects on 13C spin-lattice relaxation rates in [U-13C]nucleotides due to Mn(II), used as a substituent activating cation. The 13C relaxation measurements were performed on exclusively enzyme-bound complexes E.Mn.[U-13C]ATP and E.Mn.[U-13C]ADP at three distinct 13C NMR frequencies: 75.4, 125.7, and 181 MHz. The frequency dependence of the relaxation data has been analyzed in an effort to evaluate distances from the cation for all 10 13C nuclei in the adenosine moieties of E.Mn.ATP and E.Mn.ADP. These distance data, taken along with previously published cation-31P distances, have been used as constraints in the molecular modeling program Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations that are compatible with the distance data. It was possible to model the distances on the basis of a single enzyme-bound conformation for each of the nucleotides. The details of the enzyme-bound Mn.ATP and Mn.ADP conformations are distinguishably different from each other, indicating that structural alterations occur in the enzyme-bound reaction complex as the enzyme turns over. For example, when the adenosine moieties in the bound structures of Mn.ATP and Mn.ADP are superposed, the cation is found to be displaced by approximately 2.4 A between the two conformations, suggesting that these structural changes may involve movements with significant amplitudes. Furthermore, the NMR-determined structures of enzyme-bound Mn.ATP and Mn.ADP are significantly different from those in published X-ray crystal structures of the enzyme-nucleotide complexes.