RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE - FORMATION OF ACTIVE AND INACTIVE COMPLEXES OF PROTEINS B1 AND B2
RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE - FORMATION OF ACTIVE AND INACTIVE COMPLEXES OF PROTEINS B1 AND B2
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DOI：
10.1016/0022-2836(69)90055-2
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发表时间：
1969-01-01
影响因子：
5.6
通讯作者：
REICHARD, P
中科院分区：
文献类型：
--
作者：
BROWN, NC;REICHARD, P
核糖核苷二磷酸还原酶从大肠杆菌分离纯化过程中分为两个无催化活性的亚基，蛋白质B1和B2。当亚基混合时，酶活性很容易再生。蔗糖梯度离心表明，蛋白质B1（沉降系数7.8秒）和蛋白质B2（5.5秒）结合在镁离子的存在下，形成催化活性的复合物。在最小化解离的条件下，该复合物的沉降系数为9.7 s，由等摩尔量的两个亚基组成。酶的活性和底物特异性由作为变构效应物的核苷三磷酸调节。刺激效应物，如ATP和dTTP，并没有明显改变复合物的沉降系数。然而，在负效应dATP的存在下，在抑制酶活性的浓度下，9.7 s复合物被沉降系数为15.5 s的重物质取代。该重复合物含有等摩尔量的蛋白质B1和B2。一个类似的重复合物也形成与其他核苷三磷酸的混合物，抑制酶。另一方面，在逆转dATP抑制的浓度下，ATP阻止了重复合物的形成。我们从结果中得出结论：（1）9.7s复合物代表核糖核苷二磷酸还原酶的活性形式;（2）15.5s复合物代表酶的非活性形式;（3）两种复合物都含有等摩尔量的每个亚基。从沉降系数的差异考虑，我们建议，15.5 S复合物是9.7 S复合物的二聚体。
Ribonucleoside diphosphate reductase fromEscherichia coliB separates during purification into two catalytically inactive subunits, proteins B1 and B2. When the subunits are mixed, enzyme activity is readily regenerated. Sucrose gradient centrifugation indicated that protein B1 (sedimentation coefficient 7.8 s) and protein B2 (5.5 s) combined in the presence of magnesium ions to form a catalytically active complex. Under conditions which minimized dissociation this complex had a sedimentation coefficient of 9.7 s and consisted of equimolar amounts of the two subunits. The activity and substrate-specificity of the enzyme are modulated by nucleoside triphosphates which act as allosteric effectors. Stimulatory effectors, such as ATP and dTTP, did not appreciably change the sedimentation coefficient of the complex. However, in the presence of the negative effector dATP, at concentrations which inhibit enzyme activity, the 9.7 s complex was replaced by a heavy species with a sedimentation coefficient of 15.5 s. This heavy complex contained proteins B1 and B2 in equimolar amounts. A similar heavy complex was also formed with mixtures of other nucleoside triphosphates which inhibit the enzyme. On the other hand, the formation of the heavy complex was prevented by ATP at concentrations which reverse inhibition by dATP. We conclude from our results: (1) that the 9.7 s complex represents the active form of ribonucleoside diphosphate reductase; (2) that the 15.5 s complex represents an inactive form of the enzyme, and (3) that both complexes contain equimolar amounts of each subunit. From consideration of the differences in sedimentation coefficients we propose that the 15.5 s complex is a dimer of the 9.7 s complex.