Pyruvate Carboxylase from Bakers' Yeast

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 purilied 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 titration of this enzyme on Sephadex G-ZOO.No such correlation was observed between enzymic activity and iron content in the fractions obtained by gel liltration.Zinc was tightly bound to pyruvate carboxylase from yeast but was partially removed by gel filtration at 23" in the presence of 10 m S-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 CoClz 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 COz fixation on pyruvate.Previous studies on pyruvate carboxylase (pyruvate : COz ligase (ADP), EC 6.4.1.1)purified from chicken liver (1,Z) have