Pyruvate Carboxylase

VI. THE PRESENCE OF TIGHTLY BOUND MANGANESE

From the ¹ From the Department of Biochemistry, Western Reserve University, School of Medicine, Cleveland, Ohio 44106, and The Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

The presence of a paramagnetic metal ion in pyruvate carboxylase has been shown by the effect of this enzyme on the longitudinal relaxation rate of the protons of water as measured by pulsed nuclear magnetic resonance. The metal ion shows an enhanced effect on the proton relaxation rate (_b = 4.2) when bound to the enzyme as compared to that of the free metal ion in the presence of denatured enzyme. This observation suggested that the metal ion is manganese, and the presence of manganese in the purified enzyme is confirmed by chemical, neutron activation, emission spectral, and atomic absorption spectral analyses. Quantitative analysis by various methods has given the metal content as 2.5 to 4.3 moles of manganese per mole of enzyme or 0.6 to 1.1 moles of manganese per mole of biotin. ⁵⁴Mn is incorporated into pyruvate carboxylase in vivo. When the enzyme is isolated from chicken liver mitochondria, a good correlation is obtained between radioactivity and enzymic activity at all stages of purification subsequent to the initial mitochondrial extract. Correlation between total manganese content and enzymic activity has also been shown at various stages of purification. Studies on the ⁵⁴Mn-labeled enzyme have shown that release of ⁵⁴Mn and reduction in the enhancement of the proton relaxation rate occur only after irreversible denaturation of the enzyme. Neither the ⁵⁴Mn content nor the proton relaxation rate is affected by exposure of the enzyme to cold or ethylenediaminetetraacetate. No exchange of the bound metal ion with added manganese can be shown. Although the protein-bound manganese possesses magnetic properties consistent with divalent manganese ion, higher valency states cannot be excluded.

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