Yokota A, Harada A, Kitaoka S
Department of Agricultural Chemistry, University of Osaka Prefecture.
J Biochem. 1989 Mar;105(3):400-5. doi: 10.1093/oxfordjournals.jbchem.a122676.
An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with high specific activity (2.1 mumol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at -80 degrees 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 degrees C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 microM for CO2, 560 microM for O2, and 10.5 microM 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.
设计了一种改进方法，用于从纤细裸藻（Euglena gracilis Z）中纯化具有高比活性（2.1 μmol CO₂固定/mg蛋白质/分钟）的1,5-二磷酸核酮糖羧化酶/加氧酶（RuBisCO）。保存在-80℃的纯化酶需要用二硫苏糖醇处理才能达到完全活性。经二硫苏糖醇处理的RuBisCO被12 mM NaHCO₃和20 mM MgCl₂激活，并且在4 mM乙二胺四乙酸存在下，激活状态至少稳定60分钟。与植物酶一样，纤细裸藻酶固定的无机碳形式为CO₂。羧化酶反应至少在8分钟内随时间呈线性进行。该反应的最佳pH值为7.8至8.0。羧化酶活性随温度升高至50℃而增加。羧化反应的活化能为10.0千卡/摩尔。纤细裸藻RuBisCO对CO₂的米氏常数为30.9 μM，对O₂为560 μM，对1,5-二磷酸核酮糖为10.5 μM。根据这些酶学性质预测的光合作用速率与观察到的速率之间的数学比较表明，纤细裸藻中不存在CO₂浓缩机制。
