Eigenschaften einer Oxalacetat-Decarboxylase aus Dorschmuskulatur

Abstract

An oxaloacetic decarboxylase from cod muscle was purified 95 fold in 4 steps with 34% yield. The enzyme catalyzed the cleavage of oxaloacetate into pyruvate and CO2, as demonstrated by the stoichiometry between a direct spectrophotometric assay at 255 m[mu] ([epsilon] 255 m[mu] = 1060 for oxaloacetate), a discontinuous enzymatic assay system at 340 m[mu] for oxaloacetic and pyruvic acids, and a manometric determination of the CO2 evolved. The enzyme is not identical with other oxaloacetate metabolizing enzymes like malic enzyme, pyruvate carboxylase, or phosphoenolpyruvate carboxykinase. It is a simple, metal activated, nucleotide- and avidin-independent decarboxylase. The molecular weight was 130,000-150,000. The heat stability is remarkably high: 50% inactivation in 20 min. at 64[degree]C. The enzyme contains essential sulfhydryl groups since 5 x 10-5 M Hg2+ ions inhibit by 50%, p-chloromercuribenzoate at the same concentration by 30%. The enzymic decarboxylation of oxaloacetate follows a 1st order reaction. The activation energy amounts to 13.9 kcal/mol. The pH optimum of oxaloacetic decarboxylase was between pH 6.5 and 7.0 and the enzyme has a strict requirement for metal: Km for Mn2+ = 3.5 x 10-5 M. The Michaelis constant for oxaloacetate is 1 x 10-3 M. Oxalate exerts a strong competitive inhibition (Ki =3.5 x 10-6 M); numerous other oxo and hydroxy acids with O in the [alpha] or [beta] position inhibit much less or not at all. Rat skeletal muscle also seems to contain a highly active oxaloacetic decarboxylase.