Kurland I J, Li L, Lange A J, Correia J J, el-Maghrabi M R, Pilkis S J
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
J Biol Chem. 1993 Jul 5;268(19):14056-64.
The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.
通过在大肠杆菌中过表达肝脏和骨骼肌6-磷酸果糖-2-激酶/果糖2,6-二磷酸酶及其突变体（肝脏异构体缺失前22个氨基酸），并比较它们的动力学性质，研究了这些酶NH2末端区域的作用。肌肉异构体和缺失突变体对6-磷酸果糖的Km值分别比肝脏异构体高50倍和20倍，肝脏缺失突变体的双磷酸酶最大速度比天然肝脏异构体高4倍。肝脏异构体的磷酸化使双磷酸酶活性增加2 - 3倍，6-磷酸果糖-2-激酶对6-磷酸果糖的Km增加10 - 15倍，但尽管依赖cAMP的蛋白激酶对缺失突变体进行了等效磷酸化，这些动力学效应却大大减弱。发现骨骼肌异构体的Arg-173在功能上等同于肝脏激酶结构域中对应于必需的6-磷酸果糖结合残基的残基Arg-195。结果表明：1）肝脏和骨骼肌异构体的NH2末端区域是6-磷酸果糖亲和力的重要决定因素；2）肝脏异构体的最初22个氨基酸对双磷酸酶有抑制作用，并至少部分介导了该酶的两种活性对依赖cAMP磷酸化的反应。
