CHARACTERIZATION OF NUCLEOTIDE DEPENDANT OLIOGOMERIC STATES OF RNR1P USING SAXS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 核糖核苷酸还原酶（RNR）催化DNA复制所必需的脱氧核糖核苷酸池的产生中的限速步骤。RNR对于快速增殖的细胞至关重要，是抗HSV和抗癌药物的成功靶点。变构调节是RNR活性调节的重要方式之一，目前主要有两种模型描述RNR的变构行为。Reichard和Thelander提出的模型（RT模型）认为，底物在催化位点（c-位点）的结合受与特异性位点（s-位点）结合的给定dNTP控制，而整体酶活性取决于ATP或与活性位点结合的dATP（Eriksson，Uhlin et al. 1997）。RT模型的主要缺点之一是它不能在亚基寡聚化的背景下解释RNR的活性。Cooperman最近描述的一个全面的变构模型不仅考虑了RT模型的基本原理，而且定性地描述了酶活性如何通过核苷酸依赖性寡聚化来调节。除了s-和a-位点之外，综合模型还描述了Rnr 1中的第三个变构位点，称为六聚体位点（h-位点）。在生理上显著的浓度下，ATP与h位点的结合驱动六聚体形成。在这个模型中，dATP被认为是一种通用的抑制剂，因为无活性的四聚体形成。在这个建议中，我们希望使用小角X射线散射来表征RNR复合物的Rnr 1亚基的核苷酸依赖性寡聚化状态。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in production of the pool of deoxyribonucleotides necessary for DNA replication. Crucial for rapidly proliferating cells, RNR is a successful target for anti-HSV and anticancer drugs. Allosteric regulation consitutes one of the important modes of regualtion of RNR activity.Currently, there are two major models that describe the allosteric behavior of RNR. The model proposed by Reichard and Thelander (RT model) accounts that the substrate binding at the catalytic site (c-site) is governed by a given dNTP bound to the specificity site (s-site), while global enzyme activity is dependent on either ATP or dATP binding to the activity site(Eriksson, Uhlin et al. 1997). One of the major drawbacks of the RT model is its inability of explaining RNR's activity in the context of subunit olgiomerization. A recently described comprehensive allosteric model by Cooperman not only takes in the basic tenets of the RT model but also qualitatively describes how enzyme activity is regulated by nucleotide dependant oligomerization. In addition to the s- and a-sites, the comprehensive model describes a third allosteric site in Rnr1 known as the hexameric site (h-site). At physiologically significant concentrations, binding of ATP to h-site drives hexamer formation. In this model, dATP is regarded as a universal inhibitor because of inactive tetramer formation. In this proposal we hope to characterize the nucleotide dependant oligomerization state of Rnr1 subunit of the RNR complex using small angle X-ray scattering.