Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum.
The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.
Heda
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GD
Department of Microbiology, Southern Illinois University, Carbondale 62901.
Madigan
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MT
D002499:Centrifugation, Density Gradient; D002844:Chromatium / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006358:Hot Temperature; D005779:Immunodiffusion; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification* / Q000378:metabolism
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D012273:Ribulose-Bisphosphate Carboxylase