Pyruvate Carboxylase from Chicken Liver: MAGNETIC RESONANCE STUDIES OF THE EFFECT OF SUBSTRATES AND INHIBITORS ON THE ENVIRONMENT OF THE BOUND MANAGANESE

Abstract

Abstract Electron paramagnetic resonance spectra have been recorded for solutions of pyruvate carboxylase from chicken liver in the presence and absence of various ligands. The EPR spectra show that the intrinsic Mn(II) of the enzyme occupies a site of moderate electronic asymmetry. The symmetry of the coordination sphere of this Mn(II) is not influenced upon formation of complexes with substrates and inhibitors of the transcarboxylation partial reaction catalyzed by this enzyme. However, the electron spin relaxation time of the Mn(II), as reflected in the widths of the spectral lines, is very sensitive to the presence of substrates and inhibitors of the transcarboxylation partial reaction. Formation of enzyme-substrate or enzyme-inhibitor complexes causes a narrowing of individual lines in the spectrum, with oxalate and fluoropyruvate giving the greatest effect. Changes in the line widths of signals in the EPR spectrum of the bound Mn(II) correlate with changes in the nuclear spin relaxation time of solvent protons observed on formation of these complexes (Mildvan, A.S., Scrutton, M.C., and Utter, M.F. (1966) J. Biol. Chem. 241, 3488–3498). Both effects appear to relate to the degree of accessibility of bulk solvent to the vicinity of the bound Mn(II) with narrower EPR signals, and longer relaxation times for solvent protons, indicating decreased solvent accessibility to the Mn(II). Interaction of the biotinyl residues of pyruvate carboxylase with avidin causes broadening of the EPR spectra for the enzyme and its various complexes in accord with the prior observation of shortened solvent relaxation times in these complexes (Scrutton, M.C., and Mildvan, A.S. (1968) Biochemistry 7, 1490–1505). Thus, binding of avidin to pyruvate carboxylase appears to facilitate entry of bulk solvent to the vicinity of the Mn(II). Paramagnetic effects on the nuclear spin relaxation rates for the methyl protons of pyruvate have been measured at two frequencies. The data indicate that in the pyruvate carboxylase-pyruvate complex the methyl protons of this substrate are separated by 6 to 7 A from the bound Mn(II). The dissociation constant for the enzyme-pyruvate complex was estimated at 4 to 7 mm by equilibrium dialysis experiments, thus eliminating the possible presence of more tightly bound pyruvate molecules which would exchange slowly on the time scale of the nuclear magnetic resonance measurements. The EPR and NMR measurements lead to the conclusion that substrates and inhibitors of pyruvate carboxylase are bound near to the site of the Mn(II), but the data fail to support the previously suggested direct binding of these molecules to the Mn(II) (Mildvan, A.S., and Scrutton, M.C. (1967) Biochemistry 6, 2978–2994).