Reddy G P, Singh A, Stafford M E, Mathews C K
Proc Natl Acad Sci U S A. 1977 Aug;74(8):3152-6. doi: 10.1073/pnas.74.8.3152.
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 one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-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, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas 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.
DNA前体合成的酶是否被组织成超分子结构？该方法涉及对感染大肠杆菌的粗裂解物中几种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（dCMP羟甲基化酶、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，但不包括二氢叶酸还原酶或DNA聚合酶）的三分之一到二分之一沉降速度比根据分子量预期的要快得多。已知参与T4 DNA前体合成的宿主细胞核苷二磷酸激酶约5%与这些活性一起共沉降。为了表明这种快速沉降的物质代表一种有组织的酶复合物而不是非特异性聚集体，我们以dUMP为初始底物研究了dTTP形成的动力学。当由聚集体中的酶催化时，这个三步反应序列在几秒钟内达到最大速率，而未复合酶的等效混合物在dTTP合成达到最大速率之前需要近20分钟。聚集的作用显然是减少中间体可自由扩散的体积。因为有理由相信细胞内存在DNA前体的浓度梯度，这种酶聚集体在体外的特性可能有助于解释这种梯度是如何维持的。
