Purification, quaternary structure, composition, and properties of D-ribulose-1,5-bisphosphate carboxylase from Thiobacillus intermedius

Abstract

D-Ribulose-1,5-bisphosphate (RuBP) carboxylase was purified from glutamate-CO2-S2O32--grown T. intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2-0.8 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 EM observations of negatively stained preparations. The MW 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 MW of 54,500 .+-. 5450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The .**GRAPHIC**. [sedimentation coefficient] of the enzyme was 18.07S .+-. 0.22. EM 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 .mu.mol of RuBP-dependent CO2 fixed/min/mg of protein (at pH 8 and 30.degree. C), and the turnover number in terms of moles of CO2 fixed/mol of catalytic site per s was 2.6. The enzyme was stable for 3 mo. at -20.degree. C and at least 4 wk at 0.degree. 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-.