Catalytic Properties of Phosphopyruvate Carboxylase from Bakers' Yeast

Abstract

PHOSPHOPYRUVATE (P–EP) carboxylase (systematic name: ATP: oxalacetate carboxy-lyase) (transphosphorylating) is the enzyme responsible for carbon dioxide fixation and oxalacetic acid (OA) synthesis in bakers' yeast¹. The enzyme can be extracted soluble from acetone-dried yeast and purified with the procedure summarized in Table 1. P–EP carboxylation was measured at 30° with a spectro-photometrie method. The reaction mixture was made of 0.7 mM P–EP, 0.36 mM ADP, 3.3 mM MnCl₂, 8.3 mM NaHCO₃, 0.33 mM NADH₂, 0.05 M borate–succinate buffer pH. 5.4, malic dehydrogenase (excess) and enzyme. Total vol.: 3 ml. Variation of absorbancy at 340 mµ was read in a Beckman DU spectrophotometer at 10-sec intervals after addition of P–EP. OA decarboxylation was measured with a manometric method. Samples contained 13.3 mM OA, 0.2 mM ATP, 0.1 M borate–succinate buffer pH. 5.4 and enzyme. Total vol., 3.0 ml. The incubation was carried out at 30° for 15 min in Warburg vessels.