Purification, quaternary structure, composition, and properties of D-ribulose-1,5-bisphosphate carboxylase from Thiobacillus intermedius.
D-Ribulose-1,5-bisphosphate (RuBP) carboxylase has been purified from glutamate-CO2-S2O3(2)-grown Thiobacillus intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2 to 0.8 M sucrose gradient. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels of several acrylamide concentrations, sedimentation velocity and equilibrium measurements, and electron microscopic observations of negatively stained preparations. The molecular weights of the enzyme determined by sedimentation equilibrium and light-scattering measurements averaged 462,500 +/- 13,000. The enzyme consisted of closely similar or identical polypeptide chains of a molecular weight of 54,500 +/- 5,450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The S(0)20,w of the enzyme was 18.07S +/- 0.22. Electron microscopic examination suggested that the octomeric enzyme (inferred from the molecular measurements mentioned) had a cubical structure. The specific activity of the enzyme was 2.76 mumol of RuBP-dependent CO2 fixed/min per mg of protein (at pH 8 and 30 C), and the turnover number in terms of moles of CO2 fixed per mole of catalytic site per second was 2.6. The enzyme was stable for 3 months at -20 C and at least 4 weeks at 0 C. The apparent Km for CO2 was 0.75 mM, and Km values for RuBP and Mg2+ were 0.076 and 3.6 mM, respectively. Dialyzed enzyme could be fully reactivated by the addition of 20 mM Mg2+ and partially reactivated by 20 mM Co2+, but Cd2+, Mn2+, Ca2+, and Zn2+ had no effect. The compound 6-phosphogluconate was a linear competitive inhibitor with respect to RuBP when it had been preincubated with enzyme, Mg2+, and HCO3-.
D000596:Amino Acids / Q000032:analysis; D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000302:isolation & purification; D002474:Cell-Free System; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005971:Glutamates / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D008970:Molecular Weight; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D013855:Thiobacillus / Q000201:enzymology* / Q000378:metabolism; D013885:Thiosulfates / Q000378:metabolism
D000596:Amino Acids; D005971:Glutamates; D013885:Thiosulfates; D002245:Carbon Dioxide; D004492:Edetic Acid; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D004229:Dithiothreitol