The Magnesium Ion (Mg2+) Activation of Sheep Kidney Pyruvate Carboxylase

A detailed investigation of the Mg2+ activation of sheep kidney pyruvate carboxylase has been carried out. An analysis of the initial velocity kinetic data using two independent methods shows that the enzyme has an absolute requirement for Mg2+. Mg2+ and MgATP2- combine with the enzyme (E) in a random manner to form the final activated complex consisting of Mg2+-E-MgATP2-. Excess ATP4- is inhibitory because it shifts the equilibrium from the active complex Mg2+-E-MgATP2- to inactive complexes, e.g. Mg2+-E-ATP4-, which cannot form products. Furthermore, the analysis showed that the complex E-MgATP2- cannot break down to yield products. Investigation of the partial reactions by isotopic exchange confirmed that the presence of Mg2+ influences the binding of MgATP2- to the enzyme and has no influence on the second partial reaction, i.e. the carboxylation of pyruvate by the E-CO2 complex. The fact that Mg2+ altered the apparent Km value for MgATP2- suggested that the divalent cation induced a conformational change in the enzyme. Evidence supporting this hypothesis was obtained by determining the entropy change (ΔS° = 58.6 entropic units per mole) induced in the enzyme and by the change in emission intensity of 1-anilinonaphthalene-8-sulfonic acid when Mg2+ was bound to the enzyme.