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Pyruvate Carboxylase from Bakers' Yeast

THE PRESENCE OF BOUND ZINC

From the ¹ From the Departments of Biochemistry, Rutgers Medical School, Rutgers University, The State University of New Jersey, New Brunswick, New Jersey 08903, Northwestern University School of Medicine, Chicago, Illinois 60611, and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Pyruvate carboxylase purified from bakers' yeast was analyzed for its content of 11 metal ions. After removal of extraneous metal ions, only zinc (3.1 g atoms per mole (600,000 g) of enzyme) and iron (1.2 g atoms per mole of enzyme) were present in significant concentration, as compared with the biotin content (3.3 moles per mole). The equimolar ratio between zinc and biotin content was confirmed for four preparations of the purified enzyme. Correlation of catalytic activity, zinc, and ³H-biotin contents was demonstrated in fractions obtained by chromatography of pyruvate carboxylase from yeast on Sagarose 8F and by gel filtration of this enzyme on Sephadex G-200. No such correlation was observed between enzymic activity and iron content in the fractions obtained by gel filtration. Zinc was tightly bound to pyruvate carboxylase from yeast but was partially removed by gel filtration at 23° in the presence of 10 mm 8-hydroxyquinoline-5-sulfonate. The loss of the bound zinc under these conditions was associated with an equivalent decrease in catalytic activity.

Pyruvate carboxylase purified from yeast grown in the presence of 2 mm CoCl₂ contained approximately equal amounts of bound cobalt and zinc in a combined stoichiometry equivalent to the biotin content. The bound cobalt was removed on gel filtration in the presence of ethylenediaminetetraacetic acid, and a concomitant decrease in specific activity was observed under these conditions.

The data presented indicate that pyruvate carboxylase from bakers' yeast contains zinc as its bound metal and suggest an essential role for this bound metal ion in the catalysis of CO₂ fixation on pyruvate.

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