DEOXYNUCLEOSIDE KINASES--STRUCTURAL BASIS OF ACTIVITY

脱氧核苷激酶——活性的结构基础

• 批准号: 2187151
• 负责人: DAVID H IVES
• 金额: $ 16.63万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187151
• 关键词: active sites; bacterial proteins; circular dichroism; enzyme activity; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; fluorescent dye /probe; high performance liquid chromatography; molecular cloning; nucleoside analog; nucleotide analog; phosphotransferases; polymerase chain reaction; protein sequence; site directed mutagenesis; transfection

The long-term goal of this laboratory is to gain an understanding of catalytic and regulatory mechanisms governing the deoxynucleoside kinases in humans and various other species. A system of four deoxynucleoside kinases from Lactobacillus acidophilus R26, an organism originally found in human intestinal flora, presents an ideal model system for these studies. Whereas the human enzyme, deoxycytidine kinase, has only limited specificity, the bacterial deoxynucleoside kinases exhibit almost absolute specificity for each of the four deoxynucleoside precursors of DNA. Therefore comparison of their amino acid sequences, especially within the domains which bind substrates, should provide insight into the structural basis for nucleoside specificity. Other features of great practical interest concern the regulation of these enzymes. Each enzyme has two heterologous subunits, each bearing a different catalytic site: deoxycytidine/deoxyadenosine kinase (dCK/dAK) and deoxyquanosine/deoxyadenosine kinase (dGK/dAK), and these sites can interact mutually to stimulate enzymic activity. In addition, each site is also strongly inhibited by its own triphosphate end-product, but each end-product stimulates the opposite active site. The specific aims include: 1) High-level expression of a recently-cloned gene for DCK/DAK, paying particular attention to an unusual processing mechanism by which amino acids specified by the second and third codons of the gene are deleted from the protein product. 2) Cloning, sequencing and expression of the closely-related DGK/DAK, so that all four genetic and amino acid sequences can be compared for clues as to their substrate specificity. 3) Mapping the binding sites for substrate and end-products on each of the enzyme subunits. Reactive nucleoside and nucleotide analogs labeled with isotopes will be prepared and allowed to react with the enzymes under various conditions. Following proteolytic cleavage into fragments, the labeled peptides, presumed to represent portions of the active site, will be isolated and sequenced. In this way the amino acid sequences contributing the active site or to the end-product binding site can be identified. These experiments are also designed to test a new theory of end-product binding site can be identified. These experiments are also designed to test a new theory of end-product regulation. 4) Site-directed mutagenesis will be used to replace amino acids implicated in several enzyme functions, including substrate recognition and binding, catalysis, regulation and subunit interaction. 5) Collaborative experiments utilizing changes in fluorescence and circular dichroism will be started. The mechanisms to be examined in this model enzyme system are fundamental to many enzymes. A better understanding of how these particular enzymes work will have practical consequences, for example, in understanding how human deoxycytidine kinase can activate some chemotherapeutic agents effectively, while rejecting others.

这个实验室的长期目标是了解 脱氧核苷激酶的催化和调控机制 在人类和其他各种物种中。一种四脱氧核苷体系 嗜酸乳杆菌R26的激酶，一种最初发现的微生物 在人类肠道菌群中，提出了一种理想的模型系统 学习。而人类的酶，脱氧胞苷激酶，只有 由于特异性有限，细菌脱氧核苷酶几乎表现出 四种脱氧核苷前体的绝对特异性 DNA因此，它们的氨基酸序列的比较，特别是 在结合底物的区域内，应该提供对 核苷专一性的结构基础。卓越的其他功能 实际关注的是这些酶的调节。每种酶 有两个异源亚基，每个亚基都有不同的催化位点： 脱氧胞苷/脱氧腺苷激酶(DCK/DAK)和 脱氧鸟苷/脱氧腺苷激酶(DGK/DAK)，这些位点可以 相互作用以刺激酶的活性。此外，每个站点 也被其自身的三磷酸最终产物强烈抑制，但每个 最终产物刺激相反的活性部位。具体目标 包括： 1)新克隆的DCK/DAK基因的高效表达， 特别注意一种不寻常的处理机制，通过它 由基因的第二和第三密码子指定的氨基酸是 从蛋白质产品中删除。 2)紧密相关的DGK/DAK基因的克隆、测序和表达， 以便对所有四种基因和氨基酸序列进行比较 关于它们底物专一性的线索。 3)绘制底物和最终产物的结合部位图 酶的亚基。活性核苷及其核苷酸类似物 将制备标记有同位素的物质，并允许其与 各种条件下的酶。在蛋白水解酶裂解成 片段，标记的多肽，被推定为代表 活动地点，将被分离和排序。在这种情况下，氨基酸 贡献活性部位或最终产物结合部位的序列 可以辨认出来。这些实验也是为了测试一种新的 可以确定最终产物结合部位的理论。这些实验 也是为了测试一种新的终端产品监管理论。 4)用定点突变取代氨基酸 与多种酶功能有关，包括底物识别 以及结合、催化、调节和亚基相互作用。 5)利用荧光和荧光的变化进行协作实验 圆二向色性将被启动。 在这个模型酶系统中要研究的机制是基本的。 对许多酶都有作用。更好地了解这些特殊的酶是如何 工作将产生实际影响，例如，在理解如何 人脱氧胞苷酸激酶可激活某些化疗药物 有效地，同时拒绝其他人。