DEOXYNUCLEOSIDE KINASES--STRUCTURAL BASIS OF ACTIVITY

该实验室的长期目标是了解 脱氧核苷激酶的催化和调节机制 在人类和其他物种中。 四脱氧核苷系统 来自嗜酸乳杆菌R26的激酶， 在人类肠道植物群中，为这些提供了理想的模型系统 问题研究 而人类的脱氧胞苷激酶， 有限的特异性，细菌脱氧核苷激酶表现出几乎 对四种脱氧核苷前体中的每一种的绝对特异性 DNA. 因此，比较它们的氨基酸序列，特别是 在结合底物的领域内，应该提供对 核苷特异性的结构基础。 Great的其他特点 对这些酶的调节具有实际意义。 每种酶 具有两个异源亚基，每个亚基具有不同的催化位点： 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 相互作用以刺激酶活性。 此外，每个站点 也被其自身的三磷酸盐终产物强烈抑制，但每个 最终产物刺激相反的活性位点。 具体目标 包括以下步骤： 1)高水平表达新近克隆的DCK/DAK基因， 特别注意一种不寻常的处理机制， 由该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)DGK/DAK基因的克隆、测序及表达， 这样所有四种基因和氨基酸序列就可以进行比较， 它们的底物特异性的线索。 3)绘制每个上的底物和终产物的结合位点 酶的亚基。 反应性核苷和核苷酸类似物 将制备用同位素标记的溶液，并使其与 各种条件下的酶。 在蛋白水解裂解成 片段，标记的肽，假定代表部分的 活性位点，将被分离和测序。 这样，氨基酸 贡献活性位点或终产物结合位点的序列 可以识别。 这些实验也是为了测试一种新的 最终产物结合位点的理论可以确定。 这些实验 也是为了测试终端产品监管的新理论。 4)将使用定点突变来替换氨基酸 涉及多种酶功能，包括底物识别 以及结合、催化、调节和亚基相互作用。 5)利用荧光变化的合作实验， 将开始圆二色性。 在这个模型酶系统中要检查的机制是基本的 许多酶。 更好地理解这些特殊的酶 工作将产生实际的后果，例如，在理解如何 人脱氧胞苷激酶可激活某些化疗药物 有效地，同时拒绝其他人。

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.