DECIPHERING THE CELLULAR FUNCTION OF ALDOLASE

破译醛缩酶的细胞功能

基本信息

批准号：
6636390
负责人：
Dean R. TOLAN
金额：
$ 27.44万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-06-01 至 2005-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6636390
关键词：
X ray crystallography actins active sites aldehyde lyase binding sites chemical kinetics chimeric proteins enzyme activity enzyme structure enzyme substrate complex intein isozymes nuclear magnetic resonance spectroscopy sedimentation

项目摘要

Description: (Verbatim from the Applicant's Abstract) The long-term goals of the proposed work are to decipher the unique cellular functions of enzymes, both catalytic and "moonlighting" functions. Fructose-1,6-bisphosphate aldolase isozymes A, B, and C are crucial to metabolism in the cell, have proven to be defectivein lethal genetic diseases, and are drug targets for parasitic disease. The human isozymes share as much as 81 percent sequence identity, yet the catalytic efficiencies toward fructose-1,6-bisphosphate and fructose-1-phosphate differ by as much as two orders of magnitude between isozymes. Furthermore, aldolase isozymes have been demonstrated to bind to several important intracellular binding targets, such as F-actin and the calcium-binding S100 proteins. Evolutionary comparisons have revealed isozyme-specific residues (ISRs) and this proposal will determine which of these ISRs confer isozyme-specific functions, both in terms of their differential catalytic function and activity in protein-protein interactions. Three models will be tested: 1) binding to cellular target proteins modulates aldolase catalytic activity in an isozyme-specific manner, 2) the binding of aldolase to cellular target proteins is distinct from catalytic function and represents a "moonlighting" function of aldolase in modulating the activity of other proteins, and 3) the binding of aldolase to cellular target proteins locates aldolase (and its activities) in the cell. These models will be tested by asking: 1) can the different binding modes for substrates and enzymatic intermediates among the three aldolase isozymes be correlated to the functional differences reflected in the rate-limiting steps? 2) Do the patches of ISRs on the protein surface correlate with these functions or with moonlighting functions? This will be tested by measuring the binding interactions of aldolase isozymes to S100A1 and F-actin, and testing the same with chimeras wherein the ISR patches of one isozyme have been swapped with another isozyme. 3) What is the structure of one of the two patches of ISRs that are localized in the carboxyl-terminal region of aldolase? The proposed research will determine the complete structures of each aldolase isozyme (A, B, and C) with and without carboxyl-terminus by a combination of TROSY NMR and segmental isotopic labeling using intein-mediated protein ligation, and the substrate- specific kinetic differences and their respective rate-limiting steps by presteady-state kinetics. This work is important for understanding the roots of isozyme specificity and the interplay between catalytic and cellular functions of enzymes so critical to the field of functional genomics.

描述：（逐字研究申请人的摘要）的长期目标提出的工作是破译酶的独特细胞功能，催化和“月光”功能。果糖1,6-磷酸醛酶同工酶A，B和C对于细胞中的代谢至关重要，已被证明是有缺陷的致命遗传疾病，是寄生虫的药物靶标疾病。人类同工酶共享多达81％的序列身份，但对果糖1,6-磷酸盐和果糖-1-磷酸的差异高达两个数量级同工酶。此外，已经证明醛糖酶同工酶结合了几个重要的细胞内结合靶标，例如F-肌动蛋白和钙结合S100蛋白。进化比较揭示了同工酶特异性残基（ISRS），该提案将确定哪个这些ISR会赋予同工酶特异性功能蛋白质 - 蛋白质相互作用的差异催化功能和活性。将测试三个模型：1）与细胞靶蛋白的结合调节以同工酶特异性方式，醛酸酶催化活性，2）到细胞靶蛋白的醛糖酶不同于催化功能和代表醛酶在调节活动中的“月光”功能其他蛋白质和3）藻酶与细胞靶蛋白的结合在细胞中找到醛酶（及其活性）。这些模型将进行测试通过问：1）底物和酶促的不同结合模式可以三个醛酶同工酶之间的中间体与功能相关差异反映在限速步骤中？ 2）在上面做ISRS的补丁蛋白质表面与这些功能或月光相关功能？这将通过测量的结合相互作用来测试藻酶同工酶至S100A1和F-肌动蛋白，并用嵌合体进行相同的测试其中一个同工酶的ISR斑块已与另一个同工酶换成。 3）在本地化的ISR的两个贴片之一的结构是什么在醛酶的羧基末端区域？拟议的研究将确定每个醛酶同工酶（a，b和c）的完整结构并且不通过Trosy NMR和分段的组合而没有羧基末端使用内部介导的蛋白质连接的同位素标记，底物 - 特定的动力学差异及其各自的限制步骤 presteady state动力学。这项工作对于理解根源很重要同工酶特异性以及催化和细胞功能之间的相互作用对功能基因组学领域至关重要的酶。