Andrews T J, Ballment B
J Biol Chem. 1983 Jun 25;258(12):7514-8.
When ribulose bisphosphate carboxylase-oxygenase from Synechococcus (strain RRIMP N1) was precipitated under mildly acidic conditions, most of its small subunits remained in solution. The precipitated enzyme readily redissolved at neutral pH and remained as an octamer of large subunits with some small subunits still attached. Optimum pH for this separation was 5.3 and disulfide-reducing reagents were not necessary. The fraction of small subunits removed by a single precipitation increased with increasing NaCl concentration. Catalytic activity of small subunit-depleted enzyme was linearly proportional to the fraction of small subunits remaining, while the carboxylase:oxygenase activity ratio and the affinity for CO2 remained constant. When isolated small subunits were added back to depleted enzyme preparations at neutral pH, reassociation occurred with return of catalytic activity. Under the usual assay conditions at pH 7.7, the binding constant of the small subunits was estimated to be about 10(-9) M. The small subunits also bound avidly to surfaces. However, loss of small subunits from the enzyme during the course of purification was minimal. The results are consistent with a simple model in which only those large subunits which have a small subunit bound to them are catalytically competent. Thus, an essential, even if indirect, role for the small subunits in catalysis is indicated.
当来自聚球藻（RRIMP N1菌株）的核酮糖二磷酸羧化酶加氧酶在轻度酸性条件下沉淀时，其大部分小亚基仍留在溶液中。沉淀的酶在中性pH下很容易重新溶解，并保持为大亚基的八聚体，仍附着一些小亚基。这种分离的最适pH为5.3，不需要二硫键还原试剂。单次沉淀去除的小亚基比例随NaCl浓度的增加而增加。去除小亚基的酶的催化活性与剩余小亚基的比例呈线性比例，而羧化酶：加氧酶活性比和对CO2的亲和力保持不变。当在中性pH下将分离的小亚基重新添加到去除小亚基的酶制剂中时，会发生重新结合并恢复催化活性。在pH 7.7的常规测定条件下，小亚基的结合常数估计约为10^(-9) M。小亚基也强烈结合到表面。然而，在纯化过程中酶中小亚基的损失最小。这些结果与一个简单模型一致，即只有那些结合有小亚基的大亚基才具有催化活性。因此，表明小亚基在催化中起着重要作用，即使是间接作用。
