Zhang R, Fromm H J
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Biochemistry. 1995 Jun 27;34(25):8190-5. doi: 10.1021/bi00025a026.
Arginine 276 of porcine liver fructose-1,6-bisphosphatase (FBPase) was mutated to methionine by site-directed mutagenesis on the basis of the crystal structure of the enzyme [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., & Lipscomb, W.N. (1993) Biochemistry 32, 1844-1857]. The mutant and wild-type forms of the enzyme were purified to homogeneity and characterized by circular dichroism spectrometry (CD) and initial-rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzymes on the basis of the CD data. Replacement of Arg 276 with methionine caused a significant decrease in the enzyme's activity. The kcat for the mutant enzyme was only about 0.67% of that of the wild-type enzyme. Most importantly, the mutation caused the total loss of cooperativity for Mg2+ and changed the kinetic mechanism to one in which the substrate adds to FBPase before Mg2+ and in which all steps equilibrate rapidly relative to the conversion of the ternary complex of enzyme, substrate, and Mg2+ to products. The Ka for Mg2+ increased by only about 5-fold relative to that of the wild-type enzyme. The mutation did not change the Ki for AMP or the Hill coefficient of this allosteric inhibitor. The Ki for fructose 2,6-bisphosphate was increased by 16-fold compared with that of the wild-type enzyme. The Km for fructose 1,6-bisphosphate was similar to that of the wild-type enzyme. It is concluded that Arg 276 is critical for activity and Mg2+ cooperativity with FBPase and it determines the enzyme's kinetic mechanism.
基于猪肝果糖-1,6-二磷酸酶（FBPase）的晶体结构，通过定点诱变将该酶的第276位精氨酸突变为甲硫氨酸[张，Y.，梁，J.-Y.，黄，S.，柯，H.，&利普斯科姆，W.N.（1993年）《生物化学》32卷，1844 - 1857页]。将该酶的突变体和野生型形式纯化至同质，并通过圆二色光谱法（CD）和初速率动力学进行表征。根据CD数据，野生型和突变型酶的二级结构之间没有明显差异。用甲硫氨酸取代精氨酸276导致该酶的活性显著降低。突变型酶的kcat仅约为野生型酶的0.67%。最重要的是，该突变导致对Mg2+的协同性完全丧失，并将动力学机制改变为底物在Mg2+之前添加到FBPase上，且相对于酶、底物和Mg2+的三元复合物转化为产物的过程，所有步骤都快速达到平衡的机制。相对于野生型酶，Mg2+的Ka仅增加了约5倍。该突变没有改变AMP的Ki或这种别构抑制剂的希尔系数。与野生型酶相比，果糖2,6-二磷酸的Ki增加了16倍。果糖1,6-二磷酸的Km与野生型酶相似。得出的结论是，精氨酸276对于FBPase的活性和Mg2+协同性至关重要，并且它决定了该酶的动力学机制。
