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 结构和了解核苷二磷酸的催化机理 (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 以抑制转移以及其调节组成的能力 核苷酸池的数量。这些蛋白质的结构分析将提供一个 调节细胞功能的基础， 酶以及提供基础，以改善抗肿瘤， 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 粘球菌黄色酶的晶体结构。我们有一个 具有酶活性的酶的晶体形式。与这些 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 揭示了该酶具有一种新的核苷酸结合基序， 不同于两个预测的模式的核苷酸结合-一个 一种是基于序列分析，另一种是基于 盘基网柄藻NDF激酶的失活突变体。 NDP激酶通过乒乓机制起作用，其中， 形成自磷酸化的酶中间体。的中间体 酶促机制是磷酸组氨酸。其结构表明， 这种磷酸化的机制。虽然许多酶形成 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 对于X射线晶体学数据收集来说足够中间。 基于结构信息，我们正在研究活性位点的作用 突变体的定点诱变和稳态酶动力学。 我们将确定NDP激酶复合物的三维结构， 12种不同的底物和抑制剂NDP的这些研究 激酶-底物复合物将提供合理的结构基础 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Nm 23是一些肿瘤转移的抑制因子， 细胞类型。我们已经获得了人NDP激酶Nm 23-H2的晶体。在 除了其酶活性外，Nm 23-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因子。 这些晶体的分辨率极限为3.0埃。我们将 通过分子置换确定人体酶的结构 技术使用我们的M. xanthus酶我们还将 确定Nm 23-H2与双链 寡核苷酸。