Reinigung und Eigenschaften von Phosphoenolpyruvat-Carboxylase aus Euglena gracilis

Abstract

Dark fixations of carbon dioxide are essential reactions for plant metabolism since they supply additional carbon for synthetic processes. There exists a considerable amount of evidence, that in Euglena it is phosphoenolpyruvate carboxylase which may serve as an anaplerotic enzyme primarily responsible for the replenishment of oxaloacetate, which is drained off from the tri- carboxylic acid cycle for synthetic purposes. Phosphoenolpyruvate carboxylase has been purified approximately 420-fold from a cell-free extract of Euglena gracilis. The starting material had a specific activity of 0.085 U (μMoles phosphoenolpyruvate carboxylated per min per mg of protein). During purification a specific activity of 35 U was achieved. With respect to the carboxylase activity of the original extract, a yield of 10% was obtained. The purification procedure involved fractionation with ammonium sulphate, elimination of nucleic acids by precipitation with protamine sulphate, adsorption of the enzyme on calcium phosphate gel and desorption with increasing concentrations of phosphate buffer and two column chromatographic steps. First the enzyme was chromatographed on a DEAE-cellulose column. Elution was done with a linear gradient of KC1 in Tris-mercaptoethanol-EDTA buffer, pH 7.5. Active fractions were pooled and rechromatographed on DEAE-Sephadex A 50 in a similar manner using a linear gradient of KCl. When stored at 4°, the enzyme lost about 15% of its activity within two weeks, at −18° it was stable for a t least 4 months. The preparation was free from detectable amounts of interfering enzymes (malate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxykinase, pyruvate kinase, enolase, phosphatases, malic enzyme, glyceraldehydephosphate dehydrogenases, phosphoglycerate kinase, acetyl-CoA carboxylase, pyruvate dehydrogenase, pyrophosphatase). Phosphoenolpyruvate carboxylase of Euglena tested in Tris or phosphate buffer is maximally active a t pH 7.8. Activity is greatly stimulated on addition of sulfhydryl compounds. The enzyme is reversibly inhibited by pCMB, and the activity is nearly completely dependent on divalent cations. Mn++ and Co++ are 39% and 34% as effective as equimolar amounts of Mg++. Cu++, Ca++, Zn++, Ni++, and Fe++ have an inhibitory effect. The rate concentration data for Mg++ yield a Km value of 7.3 × 10−4M. Experimental data concerning the stoichiometry of the reaction show the equivalence between phosphoenolpyruvate consumption, CO2 fixation and Pi liberation. Plots of rate concentration data with phosphoenolpyruvate as substrate show no unusual deviations from a normal hyperbolic curve, which points to Michaelis-Menten kinetics of the enzyme. A Km value for phospho- enolpyruvate of 1.6 × 10−3 M has been estimated. While in the case of Escherichia coli phosphoenolpyruvate carboxylase, acetyl-CoA acts as a strong allosteric activator of the enzyme, this compound is without any effect on the enzyme of Euglena. In addition to the well-known activation of E. coli phosphoenolpyruvate carboxylase by acetyl-CoA, there exists an inhibitory effect with some members of the tricarboxylic acid cycle, the most predominant of which is malate. In 53 compounds tested, including members of the tricarboxylic acid cycle, amino acids, glycolytic intermediates and nucleotides, only oxaloacetic acid, citric acid and isocitric acid had an inhibitory effect. The respective values for Ki were: 8 × 10−3M for citrate. No effect like the concerted feedback inhibition could be detected. Phosphoenolpyruvate carboxylase of Euglena is thermolabile. Inactivation kinetics at 50° shows a biophasic characteristic. Activity drops in the first 2 min to a fixed level and then seems to be completely stable a t this temperature. The enzyme is protected against heat inactivation by citrate and isocitrate acting as metabolic control inhibitors. The results are discussed in terms of a metabolic feedback control of anaplerotic carbon dioxide fixation in Euglena by concentration-dependent signals out of the citric acid cycle.