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 结构并了解核苷双磷酸盐的催化机理 （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阐明酶的能力 抑制转移及其调节成分的能力 核苷酸池的。这些蛋白质的结构分析将提供 调节蜂窝功能的基础 酶以及提供改善抗塑性和的基础 某些核苷酸类似物的抗病毒特性。 我们最近确定了2.0 Angstrom分辨率X射线 粘膜球菌的晶体学结构。我们有一个 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盎司的分辨率数据 酶与核苷酸的复合物。复合物的结构 揭示该酶具有一种新颖的核苷酸结合基序 与核苷酸结合模式的两个预测不同 基于序列分析，并基于一个序列分析 distyostelium discoideum ndf激酶的非活性突变体。 NDP激酶通过乒乓球机制发挥作用 自磷酸化酶中间体形成。中级 酶促机制是一种磷酸组织。该结构表明 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 他们。 我们已经能够稳定NDP激酶磷酸酶 X射线晶体学数据收集足够的中间。 根据结构信息，我们正在研究主动位点的作用 通过位置定向诱变和稳态酶动力学的突变体。 我们将确定复合NDP激酶的三维结构 与12个不同的底物和抑制剂中的每一个。这些NDP的研究 激酶 - 基底络合物将为理性提供结构性基础 抗塑性和抗抗塑料的更有效核苷酸类似物的设计 病毒重要性。 人和鼠类NDP激酶NM23是某些转移的抑制剂 细胞类型。我们获得了人类NDP激酶NM23-H2的晶体。在 在其酶活性中，NM23-H2是一种DNA结合蛋白 是在体外的C-Myc癌基因的特定转录激活剂。 这些晶体衍射达到3.0埃埃斯特罗姆的分辨率极限。我们将 用分子替代确定人酶的结构 使用我们的叶thus酶结构的技术。我们也会 用双链确定NM23-H2复合物的结构 寡核苷酸。