Characterization of the separate kinase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase  

作　　者：杨齐衡 朱峥 李林 

机构地区：[1]State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

出　　处：《Progress in Natural Science:Materials International》2001年第8期20-28,共9页自然科学进展·国际材料（英文版）

基　　金：Project supported by the National Natural Science Foundation of China (Grant No. 39870187 and 39525005), Chinese Academy of Sciences (STZ-2-01 and KJ952-S1-13) and Shanghai Research Center of Life Sciences.

摘　　要：The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase consists of two distinct domains which catalyze the synthesis and hydrolysis of fructose-2, 6-bisphosphate, respectively. In this work the properties of the separate 6-phosphofructo-2-kinase domain were investigated. Purification of the expressed separate domain or isolation of this domain from purified glutathione S-transferase (GST) fusion protein with thrombin cleavage led to the loss of its kinase activity. Thus the domain in the GST-tagged form was characterized. The two forms of the domain with different lengths (amino acids 1 ~ 249 and 1 - 286) were very similar in kinetic property and could catalyze the formation of fructose-2,6-bisphosphate with a kcat 4-fold lower than that of the full-length enzyme. In addition, the domain was much more sensitive to guanidine inactivation and heat denaturation, and less stable at pH values below 7 than the full-length enzyme. The results suggest that the separate kinase domain ofThe bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase consists of two distinct domains which catalyze the synthesis and hydrolysis of fructose-2, 6-bisphosphate, respectively. In this work the properties of the separate 6-phosphofructo-2-kinase domain were investigated. Purification of the expressed separate domain or isolation of this domain from purified glutathione S-transferase (GST) fusion protein with thrombin cleavage led to the loss of its kinase activity. Thus the domain in the GST-tagged form was characterized. The two forms of the domain with different lengths (amino acids 1 ~ 249 and 1 - 286) were very similar in kinetic property and could catalyze the formation of fructose-2,6-bisphosphate with a kcat 4-fold lower than that of the full-length enzyme. In addition, the domain was much more sensitive to guanidine inactivation and heat denaturation, and less stable at pH values below 7 than the full-length enzyme. The results suggest that the separate kinase domain of the bifunctional enzyme is far less perfect in structure in the absence of the bisphosphatase domain, though it still possesses the 6-phosphofructo-2-kinase activity.

关 键 词：6-phosphofructo-2-kinase fructose-2 6-bisphosphatase glutathione S-TRANSFERASE fusion pro-tein kinetic property. 

分 类 号：Q819[生物学—生物工程]