RUI: Structure and Function in Enzymes Adapted to Extreme Cold

酶暴露于新的热环境进行选择，以保持功能参数，如催化速率和底物亲和力，在生理上适当的水平。 产生这些补偿性功能变化所需的结构修饰的量尚不清楚，但已经假设酶中的温度适应通过催化过程中移动的结构中或附近的灵活性的变化而发生。 该研究的目的是测试这一假设，重点是以下问题：随着热环境的变化，需要多少结构修饰才能保持最佳的酶功能？ 适应相同热环境的不同物种的直系同源酶是否在催化功能和底层结构上表现出相似的变化？ 不相关的酶是否会在结构上对温度变化做出类似的反应--特别是，在催化过程中与运动相关的区域是否会发生温度适应性变化？ 为了回答这些问题，三个糖酵解酶将被检查-肌肉型乳酸脱氢酶，丙酮酸激酶和醛缩酶-从两个不相关的组海洋鱼类适应极端寒冷-南极的notothenioids和北极和极地鳕鱼。 该项目将分为四个部分：首先，对于每个直系同源物，将在一定温度范围内确定动力学参数kcat（催化速率）和Km（底物亲和力）。 第二，每种酶的一级结构将从互补DNA序列推导出来。 第三，将创建每个极性直向同源物的三维结构的计算机模型，以确定极性酶直向同源物共享的氨基酸取代最有可能以温度适应性方式影响酶功能。 第四，将通过定点诱变来修饰代表性极性直向同源物，以确认那些残基在冷适应中的重要作用。 将测定突变体的Km和kcat值，以检验温度适应性结构变化发生在经历催化重要构象变化的区域中的假设。生物体在各种水平上适应温度的长期变化，从身体形状和大小的改变到生化途径功能的改变。 该项目旨在确定酶结构的变化-在生物化学过程中充当催化剂的蛋白质-如何在极冷温度下导致最佳酶功能。 将比较南极和北极鱼类的常见代谢酶，以发现这些不相关的物种是否发生了相同类型的功能适应。这项研究的一个重要目标是找到那些负责冷适应的氨基酸取代（蛋白质基本组成部分的变化），并确定不同鱼类的酶中是否发生了类似的取代。 这项研究将有助于阐明生物适应新环境时分子进化的速度和程度。

Enzymes exposed to novel thermal environments undergo selection to maintain functional parameters, such as catalytic rate and substrate affinity, at physiologically appropriate levels. The amount of structural modification necessary to produce these compensatory functional changes is unclear, but it has been hypothesized that temperature adaptation in enzymes takes place via changes in flexibility in or near structures that move during catalysis. The objective of the research is to test this hypothesis, focusing on the following questions: As the thermal environment changes, how much structural modification is necessary to maintain optimal enzyme function? Do orthologous enzymes from different species adapted to the same thermal environment show similar changes in catalytic function, and in underlying structure? Do unrelated enzymes show analogous changes in structure in response to temperature change - specifically, do temperature-adaptive changes occur in areas associated with movement during catalysis? To answer these questions, three glycolytic enzymes will be examined - muscle-type lactate dehydrogenase, pyruvate kinase and aldolase - from two unrelated groups of marine fishes adapted to extreme cold - the Antarctic notothenioids and the Arctic and Polar cods. The project will be divided into four segments: First, for each ortholog, the kinetic parameters kcat (catalytic rate) and Km (substrate affinity) will be determined across a range of temperatures. Second, the primary structure of each enzyme will be deduced from complementary DNA sequences. Third, computer models of the three-dimensional structures of each of the polar orthologs will be created, in order to determine which amino acid substitutions shared by the polar enzyme orthologs are most likely to affect enzyme function in a temperature-adaptive manner. Fourth, representative polar orthologs will be modified through site-directed mutagenesis to confirm the role of those residues important in cold adaptation. Km and kcat values of the mutants will be determined to test the hypothesis that temperature-adaptive structural changes occur in areas undergoing catalytically important conformational changes. Organisms adapt to long-term changes in temperature at a variety of levels, from alterations in body shape and size to modifications in the functioning of biochemical pathways. This project is designed to determine how changes in the structure of enzymes - proteins that act as catalysts in biochemical processes - lead to optimal enzyme function at extremely cold temperatures. Common metabolic enzymes from Antarctic and Arctic fishes will be compared to discover whether the same types of functional adaptation have occurred in these unrelated species. An important goal of this research is to find those amino acid substitutions (changes in the basic building blocks of the proteins) responsible for cold adaptation, and to determine whether similar substitutions have occurred in the enzymes of the different fish species. The research will help clarify the rate and extent of molecular evolution as organisms adapt to novel environments.