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 核苷二磷酸的结构并了解其催化机制 (NDF) 激酶，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 核苷酸库。这些蛋白质的结构分析将提供 调节细胞功能的基础 酶并为提高抗肿瘤和抗肿瘤作用提供基础 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 具有酶活性的酶的晶体形式。有了这些 晶体，我们已经能够收集 1.7 埃分辨率的数据 酶与核苷酸的复合物。配合物的结构 揭示该酶具有一个新颖的核苷酸结合基序，该基序非常 与核苷酸结合模式的两种预测不同 - 一种 基于序列分析和基于结构的分析 盘基网柄菌 NDF 激酶的失活突变体。 NDP 激酶通过乒乓机制发挥作用，其中 形成自磷酸化酶中间体。的中间体为 酶促机制是磷酸组氨酸。该结构表明 这种磷酸化的机制。尽管许多酶形成 磷酸组氨酸中间体，尚未确定任何结构 其中。 我们已经能够稳定 NDP 激酶磷酸酶 足以进行 X 射线晶体学数据收集的中间体。 根据结构信息，我们正在研究活性位点的作用 通过定点诱变和稳态酶动力学产生突变体。 我们将确定NDP激酶复合物的三维结构 具有 12 种不同底物和抑制剂中的每一种。新民主党的这些研究 激酶-底物复合物将为合理的 设计更有效的抗肿瘤和抗肿瘤的核苷酸类似物 病毒的重要性。 人和鼠 NDP 激酶 Nm23 是某些肿瘤转移的抑制因子 细胞类型。我们获得了人类 NDP 激酶 Nm23-H2 的晶体。在 除了其酶活性外，Nm23-H2 是一种 DNA 结合蛋白， 是体外 c-myc 癌基因的特异性转录激活剂。 这些晶体的衍射分辨率极限为 3.0 埃。我们将 通过分子置换确定人体酶的结构 使用我们的 M. xanthus 酶结构的技术。我们还将 确定具有双链的 Nm23-H2 复合物的结构 寡核苷酸。