Allosteric citrate synthase

柠檬酸合成酶在几乎所有生物体的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，这种酶就会尽可能快地催化其反应，而不会有刹车机制。在革兰氏阴性细菌这一大类细菌中，情况更为复杂：在某些条件下，它可以被生物还原剂NADH抑制(完全关闭)。为了使这一调控成为可能，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，也可以以第二种形式存在，它与NADH相互作用而不具有活性。我们已经能够使用X射线结晶学的方法来确定NADH结合或非活性形式的革兰氏阴性柠檬酸合成酶的完整结构。在目前的研究中，我们希望用分子生物学的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这样一个事实，即革兰氏阴性的柠檬酸合成酶可以被关闭，而人类的酶不能被关闭，以设计新的抗菌药物。一些最危险的细菌病原体(如与某些食物和水中毒有关的大肠埃希菌，以及与囊性纤维化肺部感染有关的铜绿假单胞菌)都是革兰氏阴性。我们正在使用高通量的化学文库筛选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所做的那样，然后改进这些发现，希望生产出能够在感染情况下阻止革兰氏阴性细菌生长的抗菌剂，而不会对人类的酶产生影响。

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.