Mechanism-Based Probes of Thiamin-Dependent Enzymes

基于机制的硫胺素依赖性酶探针

• 批准号: 8709758
• 负责人: Frank Jordan
• 金额: $ 19.08万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-10-15 至 1991-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8709758&HistoricalAwards=false
• 关键词: Mechanism; Based; Probes; Thiamin; Dependent

The long term goal of this proposal is the elucidation of the structure and mechanism of action of thiamin diphosphate- dependent pyruvate decarboxylating enzymes; pyruvate decarboxylase and pyruvate dehydrogenase complex. The latter enzyme is at a key junction in metabolism; a thorough understanding of its structure and mechanism is of fundamental biochemical importance. The enzymes will be purified in part by immunoaffinity chromatographic methods. The specific objectives are to: 1. design and synthesize mechanism-based inhibitors identify the sequence of the coenzyme and substrate binding sites and determine the location (including surrounding sequence) of the key Cys SH's of the enzymes. 2. determine the structure of the key enzyme-bound enamine using electronic spectroscopy. Ascertain its reactivity vis-a-vis protonation, disulfides, and flavin, in reactions mimicking all the important classes of thiamin diphosphate-dependent alpha-keto acid decarboxylating enzymes.

本提案的长期目标是阐明 二磷酸硫胺素结构和作用机制 依赖性丙酮酸脱羧酶 脱羧酶和丙酮酸脱氢酶复合物。 后者 酶在新陈代谢中处于一个关键的连接点;一个彻底的 了解其结构和机制， 生物化学的重要性。 这些酶将 通过免疫亲和层析方法部分纯化。 具体目标是： 1. 设计和合成基于机理的抑制剂 辅酶和底物结合位点的序列， 确定的位置（包括周围的序列） 酶的关键半胱氨酸巯基 2. 确定关键酶结合烯胺的结构 使用电子光谱学。 确定其反应性 维斯质子化、二硫化物和黄素反应 模仿所有重要的硫胺素 二磷酸依赖性α-酮酸脱羧 内切酶