DEOXYNUCLEOSIDE KINASES--STRUCTURAL BASIS OF ACTIVITY

脱氧核苷激酶——活性的结构基础

• 批准号: 3308792
• 负责人: DAVID H IVES
• 金额: $ 17.09万
• 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/3308792
• 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