Spectral Properties of Co ( II ) - Muscle Pyruvate Kinase * and Ni ( II ) - activated Rabbit

and optical properties of rabbit muscle pyruvate kinase activated with Co(II), Ni(II), Mg(II), and Mn(I1) were studied. The stoichiometry of metal binding to enzyme was found to be 4 metal ions per tetrameric enzyme for Co(I1) and Ni(I1) by carrying out circular dichroic titrations. Cu(I1) and Fe(I1) were inactive. Ca(I1) and Zn(I1) were not activating, and were inhibitory with respect to all of the active cations. The temperature dependence of the optimal velocity is similar for all activating metals. The pH rate profiles suggest that there are two classes of enzyme activation by metal ions. Mg(I1) and Mn(I1) are quite similar to each other while Co(I1) and Ni(I1) are different from them but similar to each other. Absorption, natural, and magnetic CD in the visible region were used to probe the environment of the activating divalent cation in Ni(II)-and Co(II)-activated pyruvate kinase and their complexes with substrates and inhibitors. Of these, Co(I1) appears to be more sensitive in the circular dichroism and absorption studies of the immediate environment of the bound metal ion. The spectroscopic features indicate that the divalent metal ion at the active site is six coordinate. For the Co(II)-pyruvate kinase complex, the binding of ATP causes small spectral changes, while the binding of non-nucleotide substrates, P-enolpyruvate and pyruvate, causes substantial changes which probably correspond to the ligand exchange on the enzyme. The CD spectra of pyruvate kinase-Co(II)-P-enolpyruvate complex are distinctly different from those of pyruvate kinase-Co(II)-pyruvate complex. The CD spectrum of pyruvate kinase-Co(II)-pyruvate was found to be pH-dependent with pK near 6.8 for the process. The spectral properties and presumably the local environment of Ni(I1) bound to pyruvate kinase is very insensitive to the addition of substrates and substrate analogs. This complex also undergoes a pH-dependent change with pK = 6.8. The CD spectrum of Co(I1) bound at the active site, was used to investigate the interaction of the monovalent cation binding site and the divalent cation binding site of rabbit muscle pyruvate kinase. Monovalent cations used were: (CH,),N+, not an activator, Li+, a weak activator; Cs+, an intermediate activator; and Rb+ and K+, strong activators. In the absence of substrate, the CD spectrum is essentially independent of the state of the monovalent cation binding site. The CD spectrum varies slightly as a function of the monovalent cation bound in the presence of pyruvate. In the presence of P-enolpyruvate there are dramatic changes in the visible CD …