STRUCTURAL STUDIES OF NUCLEOSIDE DIPHOSPHATE KINASE

核苷二磷酸激酶的结构研究

• 批准号: 2143692
• 负责人: ROGER L WILLIAMS
• 金额: $ 5.46万
• 依托单位: MEDICAL RESEARCH COUNCIL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-15 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143692
• 关键词: X ray crystallography; adenosine diphosphate; adenosine monophosphate; adenosine triphosphate; antineoplastics; antiviral agents; computer simulation; crystallization; cytosine nucleotides; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; guanosine diphosphate; guanosine monophosphate; nucleoside diphosphate kinase; nucleotide analog; phosphoproteins; phosphorylation; site directed mutagenesis

The broad long-term objective of the proposed work is to determine the structure and understand the catalytic mechanism of nucleoside diphosphate (NDF) kinase, an enzyme that plays a key role in nucleotide metabolism. Exciting new research is beginning to shed light on the enzyme's ability to suppress metastasis as well as its ability to regulate the composition of nucleotide pools. Structural analyses of these proteins will provide a foundation for modulating the cellular functions incumbent upon this enzyme as well as providing a basis for improving the anti-neoplastic and anti-viral properties of some nucleotide analogs. We have recently determined the 2.0 Angstrom resolution X-ray crystallographic structure of the Myxococcus xanthus enzyme. We have a crystal form of the enzyme that is enzymatically active. With these crystals, we have been able to collect 1.7 Angstrom resolution data for complexes of the enzyme with nucleotides. The structures of the complexes reveal that the enzyme has a novel nucleotide binding motif that is quite different than two predictions for the mode of nucleotide binding-one based upon the sequence analysis and one based upon the structure of an inactive mutant of the Dictyostelium discoideum NDF kinase. The NDP kinases function via a ping-pong mechanism in which an autophosphorylated enzyme intermediate is formed. The intermediate of the enzymatic mechanism is a phosphohistidine. The structure suggests a mechanism for this phosphorylation. Though many enzymes form phosphohistidine intermediates, no structure has been determined for any of them. We have been able to stabilize the NDP kinase phosphoenzyme intermediate sufficiently for X-ray crystallographic data collection. Based on structural information, we are examining the roles of active-site mutants by site-directed mutagenesis and steady-state enzyme kinetics. We will determine the three-dimensional structures of NDP kinase complexed with each of 12 different substrates and inhibitors. These studies of NDP kinase-substrate complexes will provide a structural basis for rational design of more effective nucleotide analogs of anti-neoplastic and anti- viral importance. Human and murine NDP kinase Nm23 is a suppressor of metastasis in some cell types. We have obtained crystals of a human NDP kinase, Nm23-H2. In addition to its enzymatic activity, Nm23-H2 is a DNA-binding protein that is a specific transcriptional activator of the c-myc oncogene in vitro. These crystals diffract to a resolution limit of 3.0 Angstrom. We will determine the structure of the human enzyme with molecular replacement techniques using our structure of the M. xanthus enzyme. We will also determine the structure of a complex of Nm23-H2 with a double-stranded oligonucleotide.

拟议工作的广泛长期目标是确定 二磷酸核苷的结构及其催化机理的研究 脱氧核糖核酸酶(NDF)是一种在核苷酸代谢中起关键作用的酶。 令人兴奋的新研究开始揭示这种酶的能力 抑制转移及其调节成分的能力 核糖核酸库。对这些蛋白质的结构分析将提供 为调节细胞功能奠定了基础 为提高抗肿瘤和抗肿瘤活性提供了基础 一些核苷酸类似物的抗病毒特性。 我们最近确定了2.0埃分辨率的X射线 黄色粘球菌酶的晶体结构。我们有一个 具有酶活性的酶的结晶形态。有了这些 晶体，我们已经能够收集1.7埃分辨率的数据 酶与核苷酸的复合体。络合物的结构 揭示了该酶具有一个新的核苷酸结合基序，该基序 与核苷酸结合模式的两种预测不同-一 基于序列分析和基于 盘基网柄菌NDF激酶的失活突变体。 NDP激酶通过乒乓球机制发挥作用，在乒乓球机制中 形成自动磷酸化的酶中间体。的中间体 酶的作用机制是磷酸组氨酸。这一结构表明， 这种磷酸化的机制。尽管有许多酶形成 磷酸组氨酸中间体，还没有确定任何结构 他们中的一员。我们已经能够稳定NDP激酶磷酸酶 中间体足够用于X射线晶体数据收集。 根据结构信息，我们正在检查活动站点的角色 定点突变和稳态酶动力学。 我们将确定NDP激酶复合体的三维结构 使用12种不同的底物和抑制剂。关于NDP的这些研究 激酶-底物复合体将为合理的 设计更有效的抗肿瘤和抗肿瘤的核苷酸类似物 病毒式重要性。 人和小鼠NDP激酶Nm23是某些肿瘤转移的抑制因子 单元类型。我们已经获得了人NDP激酶Nm23-H2的晶体。在……里面 除了它的酶活性，Nm23-H2是一种DNA结合蛋白，它可以 在体外是c-myc癌基因的特异性转录激活因子。 这些晶体的衍射分辨率极限为3.0埃。我们会 用分子置换法确定人酶的结构 使用我们的黄色微囊藻酶结构的技术。我们还将 Nm23-H2双链配合物的结构测定 寡核苷酸。