DECIPHERING THE CELLULAR FUNCTION OF ALDOLASE

描述：(逐字摘自申请人的摘要)的长期目标 拟议中的工作是为了破译酶的独特细胞功能， 既有催化作用，又有“兼职”功能。果糖-1，6-二磷酸缩醛酶 同工酶A、B和C对细胞内的新陈代谢至关重要，已被证明是 致命性遗传病，是寄生虫的药物靶标 疾病。人类同工酶的序列同源性高达81%，但 1，6-二磷酸果糖和1，6-二磷酸果糖的催化效率 果糖-1-磷酸之间的差异高达两个数量级 同工酶。 此外，醛缩酶同工酶已被证明与几个 重要的细胞内结合靶点，如F-肌动蛋白和 钙结合蛋白S100。进化比较揭示了 同工酶特异性残基(ISR)和这一提议将决定 这些ISR赋予同工酶特有的功能，无论是在它们的 蛋白质-蛋白质相互作用中的不同催化功能和活性。 将测试三个模型：1)与细胞靶蛋白结合调节 以同工酶特有的方式催化醛缩酶活性，2)结合 醛缩酶对细胞靶蛋白的作用不同于催化功能和 代表了醛缩酶的“兼职”功能，它调节了 其他蛋白质，以及3)醛缩酶与细胞靶蛋白的结合 在细胞中定位醛缩酶(及其活性)。这些型号将进行测试 通过提问：1)底物和酶的不同结合方式 三种醛缩酶同工酶之间的中间产物与功能相关 在速率限制步骤中反映的差异？2)ISR的补丁程序是否在 蛋白质表面与这些功能或兼职相关 功能？这将通过测量以下各项的绑定相互作用进行测试 S100A1和F-肌动蛋白的醛缩酶同工酶，并用嵌合体进行检测 其中一种同工酶的ISR斑块与另一种同工酶互换。 3)本地化的两个ISR补丁中的一个是什么结构 在醛缩酶的羧基末端区域？拟议的研究将 确定每种醛缩酶同工酶(A、B和C)的完整结构 和不含羧基末端的TROSY核磁共振和链段相结合 使用内含素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速步骤 稳态前动力学。这项工作对于理解 同工酶的特异性及其催化功能和细胞功能的相互作用 对功能基因组学领域至关重要的一系列酶。

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.