Purohit K, McFadden B A, Cohen A L
J Bacteriol. 1976 Jul;127(1):505-15. doi: 10.1128/jb.127.1.505-515.1976.
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-.
1,5 - 二磷酸核酮糖羧化酶已从以谷氨酸 - CO₂ - S₂O₃²⁻为培养基生长的中间硫杆菌中纯化得到。具体方法是先从高速离心上清液中沉淀出该酶，再经中间结晶，然后沉降到不连续的0.2至0.8M蔗糖梯度中。通过在几种不同丙烯酰胺浓度的聚丙烯酰胺凝胶上进行电泳、沉降速度和平衡测量以及对负染制剂的电子显微镜观察等标准判断，该酶是均一的。通过沉降平衡和光散射测量确定的该酶分子量平均为462,500 ± 13,000。在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳测定，该酶由分子量为54,500 ± 5,450的紧密相似或相同的多肽链组成。该酶的S(0)20,w为18.07S ± 0.22。电子显微镜检查表明，八聚体酶（从上述分子测量推断）具有立方结构。该酶的比活性为每毫克蛋白质每分钟2.76微摩尔依赖于1,5 - 二磷酸核酮糖的CO₂固定量（在pH 8和30℃下），以每秒每摩尔催化位点固定的CO₂摩尔数表示的周转数为2.6。该酶在 - 20℃下可稳定保存3个月，在0℃下至少可稳定保存4周。CO₂的表观Km为0.75mM，1,5 - 二磷酸核酮糖和Mg²⁺的Km值分别为0.076和3.6mM。透析后的酶加入20mM Mg²⁺可完全重新激活，加入20mM Co²⁺可部分重新激活，但Cd²⁺、Mn²⁺、Ca²⁺和Zn²⁺无影响。当6 - 磷酸葡萄糖酸与酶、Mg²⁺和HCO₃⁻预孵育时，它是1,5 - 二磷酸核酮糖的线性竞争性抑制剂。
