Phosphopyruvate Carboxylase from Bakers' Yeast

Bakers' yeast (Xaccharomyces cerevisiae) fixes carbon dioxide during the oxidation of glucose, pyruvate, acetate, and acetaldehyde, and also during glucose fermentation (I, 2).In experiments with C402, the kinetics of Cl4 distribution in the Krebs cycle intermediates, glutamate, and aspartate lead to the postulation (2) of oxaloacetate as the first product of carbon dioxide fixation, according to the Wood and Werkman reaction (3).This assumption has been confirmed by the isolation and purification of an enzyme that catalyzes carbon dioxide fixation according to Reaction 1.As will be shown in this paper, the en-Mn++ P-enolpyruvate + CO% + ADP e (1) oxaloacetate + ATP zyme is an adenosine triphosphate-oxaloacetate carboxylyase (trivial name, phosphopyruvate carboxylase) and on the basis of a preliminary communication (4) was included by Utter (5) in the group of phosphoenolpyruvate carboxykinases. MATERIALSYeast-Commercial preparations of pressed bakers' yeast were obtained starch-free from Compafiia Argentina de Levaduras.Acetone-dried yeast was prepared as described by Stoppani and Milstein (6) ; the powder must not be stored longer than 6 months; otherwise, the enzyme purification becomes difficult.Enzyme Preparations-Malic dehydrogenase was prepared from pig heart muscle as described by Ochoa (7).Aspartateglutamate transaminase was prepared according to Cohen (8), and the extract was fractionated with ammonium sulfate as described by Green, Leloir, and Nocito (9) to eliminate the alanineglutamate transaminase.To 100 ml of the extract was added ammonium sulfate (30 g/100 ml), and the precipitate was centrifuged at 0" and discarded; to the supernatant was further added ammonium sulfate (40 g/100 ml), and the precipitate was centrifuged off at 4", dissolved in 0.05 M buffer, pH 7.0, and dialyzed overnight against the same buffer at 0".Reagents-Oxaloacetic acid, protamine sulfate, and the nucleotides were purchased from Sigma Chemical Company, except UDP, kindly supplied by L. E. Leloir.Protamine sulfate was also obtained from British Drug Houses, Ltd.DEAE (0.6 mEq