Crystal structures of fructose 1,6-bisphosphatase: Mechanism of catalysis and allosteric inhibition revealed in product complexes
Crystal structures of fructose 1,6-bisphosphatase: Mechanism of catalysis and allosteric inhibition revealed in product complexes
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DOI：
10.1021/bi000574g
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发表时间：
2000-07-25
期刊：
影响因子：
2.9
通讯作者：
Honzatko, RB
中科院分区：
文献类型：
--
作者：
Choe, JY;Fromm, HJ;Honzatko, RB
果糖1，6-二磷酸酶（FBPase）的金属产物复合物的晶体结构揭示了AMP和二价阳离子之间的竞争。在AMP的存在下，Zn 2 +-产物和Mg 2 +-产物络合物具有仅存在于三个金属结合位点之一（位点1）的二价阳离子。该酶处于T-状态构象，具有残基52-72的无序环（环52-72）。在不存在AMP的情况下，酶以R-状态构象结晶，其中环52-72与活性位点相关联。在没有AMP的结构中，三个金属结合位点被Zn 2+占据，三个金属位点中的两个（位点1和2）被Mg 2+占据。显然，AMP与FBPase的关联使环52-72紊乱，其结果是从三个金属结合位点中的两个释放阳离子。在Mg 2+络合物（但不是Zn 2+络合物）中，果糖6-磷酸（F6 P）的1-OH基团与位点I处的金属配位，并且定向用于对结合的磷酸分子进行亲核攻击。提出了一种机制的正向反应，其中Asp 74和Glu 98一起生成一个氢氧阴离子的Mg 2+在网站2，然后取代F6 P协调。F6 P的1-氧上的负电荷的发展通过其与位点1处的Mg 2+的配位而稳定。
Crystal structures of metal-product complexes of fructose 1,6-bisphosphatase (FBPase) reveal competition between AMP and divalent cations. In the presence of AMP, the Zn2+-product and Mg2+-product complexes have a divalent cation present only at one of three metal binding sites (site 1). The enzyme is in the T-state conformation with a disordered loop of residues 52-72 (loop 52-72). In the absence of AMP, the enzyme crystallizes in the R-state conformation, with loop 52-72 associated with the active site. In structures without AMP, three metal-binding sites are occupied by Zn2+ and two of three metal sites (sites 1 and 2) by Mg2+. Evidently, the association of AMP with FBPase disorders loop 52-72, the consequence of which is the release of cations from two of three metal binding sites. In the Mg2+ complexes (but not the Zn2+ complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site I and is oriented for a nucleophilic attack on the bound phosphate molecule. A mechanism is presented for the forward reaction, in which Asp74 and Glu98 together generate a hydroxide anion coordinated to the Mg2+ at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg2+ at Site 1.