Li L, Yao W Z, Lange A J, Pilkis S J, Dong M Q, Yin Y, Xu G J
Shanghai Institute of Biochemistry, Academia Sinica, China.
Biochem Biophys Res Commun. 1995 Apr 26;209(3):883-93. doi: 10.1006/bbrc.1995.1581.
Chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was expressed in E. coli by using a pET3a T7 RNA polymerase-based expression system and was purified to homogeneity. The kinase and bisphosphatase of the expressed bifunctional enzyme had kinetic properties identical to those of the native chicken liver enzyme. However, the kinase activity of the chicken liver enzyme was 7-fold higher, while the bisphosphatase activity was 50 percent lower than those of the rat liver enzyme. Cys-256 of the rat liver bisphosphatase domain is not conserved in the chicken liver enzyme. A site-directed mutation was engineered at Cys-256 of the rat liver enzyme and the results indicate that the variation of this residue is not responsible for the difference in fructose-2,6-bisphosphatase activity between the rat and chicken liver enzymes. It is postulated that the difference in the kinase/bisphosphatase activity ratios of these two enzymes results from differences in their NH2-terminal regions.
利用基于pET3a T7 RNA聚合酶的表达系统，在大肠杆菌中表达鸡肝6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶，并将其纯化至同质。所表达的双功能酶的激酶和双磷酸酶具有与天然鸡肝酶相同的动力学性质。然而，鸡肝酶的激酶活性比大鼠肝酶高7倍，而双磷酸酶活性比大鼠肝酶低50%。大鼠肝双磷酸酶结构域的Cys-256在鸡肝酶中不保守。在大鼠肝酶的Cys-256处进行定点突变，结果表明该残基的变异与大鼠和鸡肝酶之间果糖-2,6-二磷酸酶活性的差异无关。据推测，这两种酶的激酶/双磷酸酶活性比率的差异是由它们的NH2末端区域的差异导致的。
