The Duel Functions of Acetyl-CoA Carboxylase

细胞膜是区分细胞内外的基本屏障。细胞膜由脂肪酸组成，与人类不同，细菌合成细胞膜所需的所有脂肪酸。 脂肪酸是由一系列酶催化的化学反应合成的。在脂肪酸合成中催化第一反应的酶是乙酰辅酶A羧化酶。 乙酰辅酶A羧化酶是由生物素羧化酶和羧基转移酶两种不同的酶组成的多功能酶。 该项目的第一个目标是试图了解生物素羧化酶的三维结构如何允许酶催化反应。例如，该酶由两个相同的亚基组成，其中每个亚基理论上可以催化反应。 基本问题是：如果每个亚基都能催化反应，为什么它会以二聚体的形式存在？ 一个正在测试的假设是，亚基不是独立的，而是通过交替它们的催化循环相互依赖。 类似于自行车踏板的上下运动，一个亚基催化反应，而另一个释放产物。 该项目的第二个和第三个目标涉及试图了解羧基转移酶的三维结构如何参与调节编码蛋白质的基因。 与生物素羧化酶相反，羧化转移酶由两个不同的亚基组成。 编码这些亚基的基因位于细菌染色体的不同位置，但细胞需要等量的每个亚基来产生功能性酶。 这里的基本问题是：细菌如何调节编码亚基的基因的表达，以获得等量的亚基？ 细菌中的羧基转移酶分子包含一个称为锌指的结构基序，它与RNA结合。 该假说是羧基转移酶通过与来自这两个基因的RNA结合来调节这两个基因的表达。 当营养不足时，羧基转移酶与RNA结合并抑制蛋白质合成。 当营养物质丰富，需要新的膜时，乙酰辅酶A酶的底物与RNA竞争结合，合成脂肪酸。 因此，羧基转移酶似乎有两个相互排斥的“决斗”功能：催化和翻译调节。研究的更广泛影响：该项目解决了酶催化，亚基相互作用和模型系统中翻译调控的基本问题。折衷的方法将提供一个详细的了解结构功能的关系，将有相当大的影响，我们认为酶的作用在生理水平上。该项目将在很大程度上依赖于不同的研究生和本科生进行实验，作为未来科学工作者使用生物化学和分子生物学的折衷工具的培训平台。 此外，由于该项目中的酶是一种羧化酶（使用二氧化碳作为底物），研究结果可能会对工程酶产生影响，以减少环境中的碳足迹。 最后，由于细胞膜是生命所必需的，植物中的乙酰辅酶A羧化酶是除草剂的合理靶标。 了解酶的结构和功能可以促进更有效和环境安全的除草剂的设计。

Membranes are the essential barriers that define and delineate the inside versus the outside of a cell. Membranes are composed of fatty acids, and in contrast to humans, bacteria synthesize all of the needed fatty acids for their cellular membranes. Fatty acids are synthesized by a series of enzyme catalyzed chemical reactions. The enzyme that catalyzes the first reaction in fatty acid synthesis is acetyl-CoA carboxylase. Acetyl-CoA carboxylase is a multifunctional enzyme composed of two different enzymes: biotin carboxylase and carboxyltransferase. The first aim of this project involves trying to understand how the three-dimensional structure of biotin carboxylase allows the enzyme to catalyze the reaction. For instance, the enzyme is composed of two identical subunits where each subunit can in theory catalyze the reaction. The fundamental question is: if each subunit is capable of catalyzing the reaction why does it exist as a dimer? One hypothesis that is being tested is that the subunits are not independent but instead depend on each other by alternating their catalytic cycles. Similar to the up and down motion of a bicycle pedal, one subunit catalyzes a reaction while the other releases products. The second and third aims of the project involve trying to understand how the three-dimensional structure of carboxyltransferase participates in regulating the genes that code for the protein. In contrast to biotin carboxylase, carboxyltransferase is composed of two different subunits. The genes coding for these subunits are located at different positions in the bacterial chromosome yet the cell requires equal amounts of each subunit to make a functional enzyme. The fundamental question here is: how does a bacterium regulate expression of the genes coding for the subunits to obtain equal amounts? The carboxyltransferase molecule in bacteria contains a structural motif called a zinc finger that binds to RNA. The hypothesis is that carboxyltransferase regulates expression of both genes by binding to the RNA derived from those genes. When nutrients are low, carboxyltransferase binds to the RNA and inhibits protein synthesis. When nutrients are abundant and new membranes are needed, the substrate for the enzyme acetyl-CoA competes with RNA for binding and fatty acids are synthesized. Thus, carboxyltransferase appears to have two mutually exclusive "duel" functions: catalysis and translational regulation. Broader Impacts of the Research: This project addresses fundamental questions in enzyme catalysis, subunit interactions, and regulation of translation in a model system. The eclectic approach will provide a detailed understanding of structure-function relationships that will have considerable impact on our view of the roles of the enzyme on the physiological level. This project will rely heavily on diverse graduate and undergraduate students to perform the experiments, serving as a training platform for the future scientific workforce using the eclectic tools of biochemistry and molecular biology. In addition, because the enzyme in this project is a carboxylase (using a form of carbon dioxide as substrate) the results of the research may have ramifications for engineering enzymes to reduce the carbon footprint in the environment. Lastly, because membranes are essential for life, acetyl-CoA carboxylase in plants is a logical target for herbicides. Understanding the structure and function of the enzyme can facilitate the design of more potent and environmentally safe herbicides.