Characterization of Ribulose 1, 5-Bisphosphate Carboxylase/Oxygenase from Euglena gracilis Z1

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Abstract
An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) with high specific activity (2.1 μmol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at −80°C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO3 and 20 mM MgCl2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the Euglena enzyme was CO2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50°C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 μM for CO2, 560 μM for O2, and 10.5 μM for ribulose 1,5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO2-concentrating mechanism in E. gracilis.