Giroux E, Williams M K, Kantrowitz E R
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167.
J Biol Chem. 1994 Dec 16;269(50):31404-9.
The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidney enzyme expressed in Escherichia coli. Compared with wild-type enzyme, kinetic parameters of the altered enzyme characterizing catalytic efficiency, magnesium binding, and inhibition by AMP differed but by less than an order of magnitude; affinity for substrate fructose 1,6-bisphosphate was 10-fold poorer, and affinity for inhibitor fructose 2,6-bisphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the enzyme due to replacement of Arg-243 by Ala and suggested that in the mutant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The differences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabolic enzyme.
猪肾果糖-1,6-二磷酸酶（EC 3.1.3.11）的活性位点位于亚基之间，一条链上的精氨酸-243与相邻链活性位点中的果糖-1,6-二磷酸或果糖-2,6-二磷酸相互作用。在本研究中，利用定点诱变技术将精氨酸-243替换为丙氨酸，并在大肠杆菌中表达克隆的猪肾酶。与野生型酶相比，改变后的酶在催化效率、镁结合以及AMP抑制方面的动力学参数有所不同，但差异小于一个数量级；对底物果糖1,6-二磷酸的亲和力降低了10倍，对抑制剂果糖2,6-二磷酸的亲和力降低了1000倍。进行了分子动力学模拟，以确定由于精氨酸-243被丙氨酸取代而导致的酶活性位点可能的变化，结果表明在突变酶中一个阳离子基团的缺失导致活性位点的重组，尤其涉及赖氨酸残基269和274。突变酶性质的差异表明精氨酸-243在果糖-1,6-二磷酸酶功能中至关重要，并在功能基础上证实了这种重要代谢酶中共享的活性位点。
