Composition, quaternary structure, and catalytic properties of D-ribulose-1, 5-bisphosphate carboxylase from Euglena gracilis.
D-Ribulose-1,5-bisphosphate carboxylase has been purified in one step by sedimenting extracts of autotrophically-grown Euglena gracilis into a linear 0.2-0.8 M sucrose density gradient. The resultant product was pure by the criteria of disc electrophoresis in gels polymerized from 5 or 7.5% acrylamide and sedimentation. The molecular weight of the enzyme estimated by density gradient centrifugation and electrophoresis in gels polymerized from various concentrations of acrylamide was 5.25 X 10(5). The S20,W was 16.4 S. Dissociation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate established that the enzyme was composed of two types of subunits (mr 50,000 and 15,000). The oligomeric structure was visualized through negative staining and transmission electron microscopy leading to a model for the quaternary structure. Although the enzyme was moderately unstable, the estimated maximal specific activity was 1.6 mumol CO2 fixed min-1 mg protien-1 at 30 degrees C and pH 8.0 Km values were 2.2 m M, 15. 1 MUM and 0.63 mM for Mg2+, ribulose 1,5-bisphosphate, and CO2, respectively, when measured under air. 6-Phospho-D-gluconate was a noncompetitive inhibitor with respect to ribulose 1,5-bisphosphate (Ki = 0.04 mM). Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme was also an oxygenase. The latter was confirmed by experiments showing a molar equivalence between ribulose-1,5-bisphosphate-dependent oxygen consumption and phosphoglycerate production.
D000596:Amino Acids / Q000032:analysis; D002262:Carboxy-Lyases; D002499:Centrifugation, Density Gradient; D004589:Electrophoresis, Disc; D005056:Euglena gracilis / Q000201:enzymology*; D005942:Gluconates / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D010100:Oxygen / Q000494:pharmacology; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate; D013403:Sugar Phosphates / Q000494:pharmacology; D014461:Ultracentrifugation
D000596:Amino Acids; D005942:Gluconates; D046911:Macromolecular Substances; D013403:Sugar Phosphates; D012967:Sodium Dodecyl Sulfate; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen