Nuclear magnetic relaxation studies of the conformation of adenosine 5'-triphosphate on pyruvate kinase from rabbit muscle.
The conformation of adenosine 5'-triphosphate in the manganese complex of pyruvate kinase from rabbit muscle was determined from six metal to nucleus distances derived by nuclear magnetic relaxation techniques. On the enzyme, no direct metal-ATP coordination exists. The phosphorous atoms of ATP are 4.9 to 5.1 A away from manganese, a distance which indicates either a predominantly (greater than or equal to 94%) second sphere complex or, less likely, a highly distorted inner sphere complex. Thus, water ligands or ligands from the protein might intervene between the ATP molecule and the divalent metal ion and facilitate their interaction. The metal-gammaP distance of 5 A for pyruvate kinase-bound ATP is equal to that found for the phosphorous atom of phosphoenolpyruvate and cobalt(II) on pyruvate kinase (Melamud, E., and Mildvan, A. S. (1975) J. Biol. Chem. 250, 8193-8201), which is consistent with the overlap in space of the P-enolpyruvate-phosphorus and the gammaP of ATP at the active site. This observation  explains the competitive binding of these two substrates to the enzyme, as detected by NMR and by early kinetic studies. From the phosphorus data and from measurements of the relaxation rates of 3 protons of ATP in the pyruvate kinase-metal-ATP complex, the conformation of ATP was characterized as extended with distances of 6.0, 9.1, and 7.5 A from manganese to the H8, H2, and H'1 protons, respectively. The torsion angle about the glycosidic bond (chi) which defines the conformation of the enzyme-bound riboside and adenine rings was determined to be 30 degrees. In contrast, the conformation of the binary Mn(II)-ATP complex in solution is folded around the metal with direct manganese coordination of the alpha-, beta-, and gamma-phosphorus atoms, and with metal to proton distances of 4.5, 6.4, and 6.2 A for the H8, H2, and H'1 protons, suggesting a second sphere manganese-adenine interaction. The chi angle equals 90 degrees for the binary complex primarily because of the metal-base interaction. Thus, a profound change in the conformation and structure of Mn(II)-ATP from a folded chelate to an extended second sphere complex results when the nucleotide binds to pyruvate kinase.
D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese; D008433:Mathematics; D008958:Models, Molecular; D008968:Molecular Conformation; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase; D011817:Rabbits
D008345:Manganese; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase