Protein-Protein Interactions in DNA Precursor Biosynthesis

催化连续反应的酶越来越多地被发现与细胞内有关。有时这种结合涉及多功能蛋白质，如真核脂肪酸合成酶复合物中的蛋白质，有时是紧密连接的酶复合物，如甘氨酸裂解系统，有时是膜相关复合物，如线粒体呼吸复合物。然而，大多数酶是可溶性蛋白质，容易与其他蛋白质分离。在细胞内发现的高蛋白质浓度下，这些酶可以结合成松散结合的复合物，已故的Paul Srere称之为代谢子。一种对代谢子概念的结构和动力学分析具有有利性质的复合物是噬菌体T4 dNTP合成酶复合物。该实验室先前的研究已经证明了一种复合物，其中含有至少八种噬菌体编码的蛋白质和两种宿主细胞来源的蛋白质。在孤立的复合物内的个体活动是动力学联系的。间接证据表明，该复合物与DNA复制装置相关，并且它在以每秒500-800个核苷酸的速率将脱氧核糖核苷三磷酸（dNTPs）快速递送到复制体以维持DNA链方面起着重要作用。所有已知在复合物中的酶和所有T4 DNA复制蛋白都可作为纯化的重组蛋白获得，从而允许通过研究复合物的部分重构形式来鉴定特定蛋白质-蛋白质相互作用和分析动力学偶联的多种方法。本研究的主要目的是：（1）鉴定T4 dNTP合成酶复合体中蛋白质之间的直接和间接相互作用，以及与该复合体相关的T4复制蛋白之间的相互作用。这涉及在光学生物传感器，以及蛋白质亲和层析和非变性凝胶电泳分析;（2）确定底物和变构效应蛋白质-蛋白质协会的影响。证据表明，小分子对dNTP合成酶复合物中的蛋白质缔合具有显著的影响;（3）确定蛋白质-蛋白质相互作用对单个酶的动力学行为和连接的多步反应途径的影响。部分重组复合物的动力学分析探讨了动力学耦合的本质。稳态分析，并与同事合作，使用猝灭流技术的预稳态分析正在使用;（4）为了确定dNTP合成酶复合物是否与体内复制机制相关。这些实验涉及E.大肠杆菌dNTP合成酶，特别是核糖核苷酸还原酶和胸苷酸合成酶，也正在进行合作。复合物中某些酶的晶体结构已经确定，其他结构的确定正在进行中。随着有关蛋白质和对接位点的结构信息的出现，这些数据将与本研究的酶学数据合并，试图建立这种特定代谢子的统一图像。

Enzymes catalyzing sequential reactions are increasingly found to be associated within cells. Sometimes the association involves multifunctional proteins, such as those in eukaryotic fatty acid synthetase complexes, sometimes tightly linked enzyme complexes, such as the glycine cleavage system, and sometimes membrane-associated complexes, such as mitochondrial respiratory complexes. Most enzymes, however, are soluble proteins, readily isolated free of other proteins. At the high protein concentrations found within cells, such enzymes can associate into loosely bound complexes, which the late Paul Srere termed metabolons. A complex that has favorable properties for structural and kinetic analysis of the metabolon concept is the bacteriophage T4 dNTP synthetase complex. Previous research from this laboratory has demonstrated a complex containing at least eight phage-coded proteins and two of host cell origin. Individual activities within the isolated complex are kinetically linked. Indirect evidence suggests that the complex is associated with the DNA replication apparatus and that it plays an important role in delivering deoxyribonucleoside triphosphates (dNTPs) to replisomes fast enough to sustain DNA chain at rates of 500-800 nucleotides per second. All of the enzymes known to be in the complex and all T4 DNA replication proteins are available as purified recombinant proteins, allowing numerous approaches to identifying specific protein-protein interactions and analysis of kinetic coupling through studies on partially reconstituted forms of the complex. This project includes the following specific aims: (1) To identify direct and indirect interactions among proteins in the T4 dNTP synthetase complex and interactions with T4 replication proteins shown to associate with the complex. This involves analysis in an optical biosensor, as well as protein affinity chromatography and nondenaturing gel electrophoresis; (2) To define effects of substrates and allosteric effectors upon protein-protein associations. Evidence suggests that small molecules have dramatic effects upon protein associations in the dNTP synthetase complex; (3) To define effects of protein-protein interactions upon kinetic behavior of individual enzymes and linked multi-step reaction pathways. Kinetic analysis of partially reconstituted complexes explores the nature of kinetic coupling. Steady-state analyses, and, in collaboration with a colleague, pre-steady-state analyses using quench-flow techniques are being used; (4) To ascertain whether the dNTP synthetase complex is associated with the replication machinery in vivo. These experiments involve immunolocalization of E. coli dNTP-synthesizing enzymes, specifically, ribonucleotide reductase and thymidylate synthase, and are also being done in a collaborative effort. Crystal structures of some of the enzymes in the complex have been determined and other structure determinations are under way. As structural information about proteins and docking sites emerges, these data will be merged with the enzymological data from this study, in an attempt to develop a unified picture of this particular metabolon.