Electron paramagnetic resonance studies of manganese (II)-pyruvate kinase-substrate complexes.

Electron paramagnetic resonance spectra of M&II) bound to pyruvate kinase and its substrate and inhibitor complexes are examined in solution. The binary complex of Mn(I1) with the enzyme gives a spectrum which indicates that the electronic symmetry of the bound cation is only moderately distorted upon liganding to the protein. The spectrum of the binary enzyme-Mn(I1) complex is unaltered upon formation of ternary complexes with either ADP or ATP. However, when ternary complexes are formed with either pyruvate or P-enolpyruvate, there are gross changes in the position and shape of the spectral lines which correlate with changes in the electronic symmetry of the bound Mn(I1) and the electron spin relaxation time, respectively. The former changes are indicative of changes in the ligands of the bound metal ion and the latter of the accessibility of the binding site to the bulk solvent. Minor changes are observed in the spectrum of the pyruvate complex upon addition of ATP or enzymatic enolization cofactors such as arsenate or inorganic phosphate. Preliminary studies of Mn(I1) complexes with chelating agents have revealed a striking similarity in spectra for the 2: 1 complex of iminodiacetic acid and Mn(I1) and the ternary complex of manganese-pyruvate kinase and pyruvate. Neither the spectrum of the binary complex nor that of the ternary complex with pyruvate is influenced by the presence or absence of activating monovalent cations such as K+. In contrast, spectra for the ternary complex of P-enolpyruvate are influenced by the species of monovalent cation in solution. In the presence of the nonactivating tetramethylammonium cation, the spectrum for the P-enolpyruvate ternary complex is a superposition of spectra for two distinct species. One species gives a very anisotropic spectrum which resembles that obtained in the presence of K+, whereas the