Characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis Z.
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.
Yokota
A
A
Department of Agricultural Chemistry, University of Osaka Prefecture.
Harada
A
A
Kitaoka
S
S
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D003720:Densitometry; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005056:Euglena gracilis / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D004229:Dithiothreitol