Temperature dependent conformational changes at the active site of pyruvate kinase: A 7Li nuclear magnetic resonance study

Abstract

The interaction of Li +, a weak activator of pyruvate kinase, with substrate and inhibitor complexes of the enzyme has been investigated by magnetic resonance techniques. Proton relaxation rate (PRR) titrations indicate that the dissociation constant of Li + from the ternary enzyme-Mn(II)-phospho enolpyruvate (P-enolpyruvate) complex is 15 m m at 5 °C and 17 m m at 30 °C. The electron paramagnetic resonance spectrum of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex is the superposition of spectra for two distinct species (Reed, G. H., and Cohn, M. (1973) J. Biol. Chem. 248, 6436–6442). Low temperatures favor the form giving rise to the more nearly isotropic spectrum, whereas high temperatures favor the species giving rise to the anisotropic “K +-like” spectrum. 7Li nuclear magnetic resonance data are consistent with a model in which the two forms observed by epr correspond to differing Mn(II) to Li(I) distances. The form giving rise to the anisotropic spectrum is characterized by a Mn(II) to Li(I) distance of 4.7 Å, and in the more isotropic form this distance is approximately 9 Å. The 4.7 Å separation of the Mn(II) and Li(I) in the anisotropic form of the complex compares favorably with the 4.9 Å separation of Mn(II) and T1(I) (Reuben, J., and Kayne, F. J. (1971) J. Biol. Chem. 246, 6227–6234) in the P-enolpyruvate complex, although T1 + is a much better activator of the pyruvate kinase reaction. Thus, a change in the distance between the monovalent and divalent cations does not account quantitatively for the lower activation by Li +, inasmuch as more than 50% of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex has the “active” conformation with respect to the separation of the cations and the epr spectrum of the complex. As reported previously (Reed, G. H., and Morgan, S. D. (1974) Biochemistry 13, 3537–3541), the dissociation constant of oxalate and the epr spectrum for the ternary complex of pyruvate kinase with Mn(II) and oxalate are not influenced by the species of monovalent cation present. The nuclear relaxation rates of Li + are increased in the presence of the ternary oxalate complex, although the separation of the Mn(II) and Li(I) appears to be much greater than for the “anisotropic” form of the P-enolpyruvate complex.