STRUCTURE-FUNCTION STUDIES OF YEAST FATTY ACID SYNTHASE

酵母脂肪酸合成酶的结构与功能研究

• 批准号: 3305680
• 负责人: JAMES K STOOPS
• 金额: $ 14.48万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305680
• 关键词: active sites; acyl carrier protein; analytical ultracentrifugation; chemical association; chemical kinetics; computer simulation; cryoscopy; electron microscopy; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; fatty acid biosynthesis; fatty acid synthase; gold; image processing; immunoelectron microscopy; laboratory rabbit; molecular cloning; mutant; scanning electron microscopy; site directed mutagenesis; stainings; transacylation; yeasts

Fatty acids are essential components of biological membranes in eukaryotes and in higher organisms. In these organisms, the derivatives of fatty acids serve as highly concentrated energy stores, hormones, and intracellular messengers. Moreover, in humans, fatty acids play a major role in arteriosclerosis, which can lead to heart attack and stroke. To understand fully the etiology of arteriosclerosis we must first understand fatty acid synthesis on the molecular level. In this study, the yeast fatty acid synthase will be used as a model system for fatty acid synthesis because this enzyme is well suited to a variety of physicochemical and molecular biological approaches. The yeast fatty acid synthase is a macromolecular complex (M-r = 2.4 x 10[6]) comprised of six copies of two different subunits called alpha and beta (alpha6beta6). The structural organization of the complex will be studied using different electron microscopic techniques (negative stain, cryo, and immuno) together with image processing to enhance structural details and to achieve a three-dimensional reconstruction of the complex. The six sites of fatty acid synthesis will be located using an active site-directed undecagold label by scanning electron microscopy. How the six alpha and six beta subunits (M-r equals approximately 220,000 each) self-assemble to form the active enzyme and how the functional domains are organized within the subunits will be studied by electron microscopy and analytical ultracentrifugation and by immunoelectron microscopy, respectively. An enzyme containing a truncated alpha subunit lacking the N-terminal acyl carrier protein domain will be examined to assess its role in the structure and function of the complex and to determine the role of the C-terminal portion of the alpha subunit in the total structure and overall function of the enzyme. Site-directed mutagenesis will be employed to obtain yeast synthase lacking the primer function (acetyl transacylase) and the 4'-phosphopantetheine prosthetic group to investigate the mechanism of the transacetylation pathway by enzyme kinetics.

脂肪酸是生物膜的重要组成部分， 真核生物和高等生物。 在这些生物体中， 脂肪酸作为高度集中的能量储存，激素， 细胞内信使 此外，在人类中，脂肪酸起着主要作用， 动脉硬化，可导致心脏病发作和中风。 到 充分了解动脉硬化的病因，我们必须首先 从分子水平理解脂肪酸的合成。 在本研究中， 酵母脂肪酸合酶将用作脂肪酸合成的模型系统 酸合成，因为这种酶非常适合于各种 物理化学和分子生物学方法。 酵母脂肪 酸合酶是一种大分子复合物（M-r = 2.4 × 10[6]），包括 两种不同亚基的六个拷贝，分别是alpha和beta （α 6 β 6）。 该建筑群的结构组织将是 使用不同的电子显微镜技术（负染色， 冷冻和免疫）与图像处理一起增强结构 细节，并实现了复杂的三维重建。 脂肪酸合成的六个位点将使用活性 通过扫描电子显微镜进行定点十一金标记。 如何 6个α和6个β亚基（M-r分别约等于220，000） 自组装形成活性酶以及功能结构域如何 将通过电子显微镜进行研究 和分析性超离心以及免疫电子显微镜， 分别 一种含有截短的α亚基的酶， 将检查N-末端酰基载体蛋白结构域以评估其 在复合物的结构和功能中的作用， α亚基的C-末端部分在总的 酶的结构和功能。 定点诱变 将用于获得缺乏引物功能的酵母合酶 （乙酰基转酰酶）和4 '-磷酸泛酰巯基乙胺辅基， 酶促转乙酰化途径的机理研究 动力学。