ENZYME ASSOCIATIONS IN T4 PHAGE DNA PRECURSOR SYNTHESIS
ENZYME ASSOCIATIONS IN T4 PHAGE DNA PRECURSOR SYNTHESIS
复制标题
DOI：
10.1073/pnas.74.8.3152
复制
发表时间：
1977-01-01
影响因子：
11.1
通讯作者：
MATHEWS, CK
中科院分区：
文献类型：
--
作者：
REDDY, GPV;SINGH, A;MATHEWS, CK
描述了一个直接的方法来解决这个问题：DNA前体合成的酶是否组织成超分子结构？该方法涉及对受感染大肠杆菌粗裂解物中几种 T4 噬菌体编码的早期酶活性进行沉降分析。所测试的几种活性中的三分之一到 1/2。sbd.dCMP 羟甲基酶、dTMP 合成酶、脱氧核苷 5''-单磷酸激酶、脱氧尿苷三磷酸酶，可能还有 dCMP 脱氨酶，但不是二氢叶酸还原酶或 DNA 聚合酶。sbd. 沉淀的速度比根据分子量预期的要快得多。已知大约 5% 的宿主细胞核苷二磷酸激酶参与 T4 DNA 前体合成，与这些活性共同沉积。为了表明这种快速沉淀的材料代表的是有组织的酶复合物而不是非特异性聚集体，研究了以 dUMP 作为初始底物形成 dTTP 的动力学。当由聚集体中的酶催化时，这三步反应序列在几秒钟内达到其最大速率，但未复合酶的等效混合物需要近 20 分钟才能 dTTP 合成达到其最大速率。聚集的作用显然是减少中间体自由扩散的体积。因为有理由相信 DNA 前体的细胞内浓度梯度存在，所以这种酶聚集体的体外特性可能有助于解释如何维持这种梯度。
A direct approach is described to the question: are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to 1/2 of several activities tested.sbd.dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5''-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase.sbd.sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, the kinetics of formation of dTTP with dUMP as the initial substrate was studied. This 3 step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, but an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.