Allosteric citrate synthase

变构柠檬酸合酶

• 批准号: 6862-2008
• 负责人: Duckworth, Harry
• 金额: $ 2.91万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=498156
• 关键词: Allosteric; citrate; synthase

The enzyme citrate synthase plays a central role in the production of metabolic energy in almost all organisms. In most organisms, including humans, the enzyme catalyzes its reaction as quickly as possible, with no braking mechanism as long as the raw materials are available. In one large class of bacteria, the Gram-negative bacteria, the situation is more complex: under certain conditions, it can be inhibited (shut down completely) by a biological reducing agent, NADH. To make this regulation possible, Gram-negative citrate synthase has evolved to a larger enzyme which can exist in either an active form, or in a second form which interacts with NADH and is inactive. We have been able to use the method of X-ray crystallography to determine the complete structure of Gram-negative citrate synthase in the NADH-binding or inactive form. In the present research, we wish to use molecular biology methods to force this enzyme into the active form, and determine that structure. Our second aim is to exploit the fact that Gra-negative citrate synthase can be turned off, while the human enzyme cannot, to design new antimicrobial drugs. Some of the most dangerous bacterial pathogens (like E. coli, involved in some kinds of food and water poisoning, and Pseudomonas aeruginosa, involved in lung infections in cystic fibrosis) are Gram-negatives. We are using high-throughput screening of chemical libraries to identify novel compounds that can inhibit Gram-negative citrate synthase, as NADH does, and then refine these discoveries in the hope of producing antimicrobials that can turn off Gram-negative bacterial growth in the case of infections, while having no effect on the human enzyme.

在几乎所有生物体中，柠檬酸合成酶在代谢能量的产生中起着核心作用。在包括人类在内的大多数生物体中，只要原料可用，酶就会尽可能快地催化其反应，没有制动机制。在革兰氏阴性菌这一大类细菌中，情况更为复杂：在某些条件下，它可以被一种生物还原剂NADH抑制（完全关闭）。为了使这种调节成为可能，革兰氏阴性柠檬酸合成酶已经进化成一种更大的酶，它可以以活性形式存在，也可以以与NADH相互作用且不活跃的第二种形式存在。我们已经能够使用x射线晶体学方法来确定革兰氏阴性柠檬酸合成酶在nadh结合或失活形式下的完整结构。在目前的研究中，我们希望利用分子生物学方法迫使这种酶进入活性形式，并确定其结构。我们的第二个目标是利用gra -柠檬酸合酶可以关闭的事实，而人类的酶不能，设计新的抗菌药物。一些最危险的细菌病原体（如大肠杆菌，导致某些食物和水中毒，铜绿假单胞菌，导致囊性纤维化肺部感染）是革兰氏阴性的。我们正在使用高通量筛选化学文库来识别能够抑制革兰氏阴性柠檬酸合成酶的新化合物，就像NADH一样，然后对这些发现进行改进，以期生产出能够在感染的情况下阻止革兰氏阴性细菌生长，同时对人类酶没有影响的抗菌剂。