Danson M J, Harford S, Weitzman P D
Eur J Biochem. 1979 Nov;101(2):515-21. doi: 10.1111/j.1432-1033.1979.tb19746.x.
Naturally occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a 'large' enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as Escherichia coli, also by 2-oxoglutarate. On the other hand, Gram-positive bacteria and all eukaryotes produce a 'small' citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies we have explored the possibility of genetically altering one type of citrate synthase to the other. By mutagenesis and suitable selection we have succeeded in isolating a mutant of E. coli whose citrate synthase is both 'small' and insensitive to NADH and 2-oxoglutarate. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.
天然存在的柠檬酸合酶可分为不同的分子和催化类型。革兰氏阴性菌产生一种“大型”酶，受NADH变构抑制，而在诸如大肠杆菌等兼性厌氧菌中，还受2-氧代戊二酸抑制。另一方面，革兰氏阳性菌和所有真核生物产生一种“小型”柠檬酸合酶，对这些代谢物不敏感。作为结构-功能研究的补充，我们探索了将一种类型的柠檬酸合酶通过基因改造转变为另一种类型的可能性。通过诱变和适当的筛选，我们成功分离出大肠杆菌的一个突变体，其柠檬酸合酶既是“小型”的，又对NADH和2-氧代戊二酸不敏感。文中描述了该酶的一些特性。这类突变酶为研究柠檬酸合酶、其调控及其天然多样性提供了一种新方法。
