Ko Y H, Vanni P, Munske G R, McFadden B A
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Biochemistry. 1991 Jul 30;30(30):7451-6. doi: 10.1021/bi00244a012.
The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.
在pH 6.8（37摄氏度）条件下，焦碳酸二乙酯可使来自大肠杆菌的188-kDa四聚体异柠檬酸裂解酶失活，呈现饱和动力学，同时组氨酸残基266和306会发生修饰。底物异柠檬酸、乙醛酸或乙醛酸加琥珀酸可保护该酶不被失活，但单独的琥珀酸则不能。用羟胺处理使失活的酶脱除乙氧羰基后，异柠檬酸裂解酶的活性得以恢复。目前的结果表明，基团特异性修饰试剂焦碳酸二乙酯可能在确定酶活性位点组氨酸残基的位置方面具有普遍用途。
