Feese M D, Faber H R, Bystrom C E, Pettigrew D W, Remington S J
Central Laboratories for Key Technology 1 - 13-5 Fukuura Kanazawa Yokohama 236, Japan.
Structure. 1998 Nov 15;6(11):1407-18. doi: 10.1016/s0969-2126(98)00140-3.
Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70.
We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity.
We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.
大肠杆菌的甘油激酶（GK）是一种速度调节型（V系统）酶，有三种具有独立机制的别构效应物：果糖-1,6-二磷酸（FBP）；磷酸载体蛋白IIAGlc；以及腺苷核苷酸。该酶在溶液中以功能性二聚体形式存在，二聚体可逆地缔合形成四聚体。GK是ATP酶超家族的成员，该家族共享一个共同的ATP酶结构域，并且被认为在其催化循环的一个内在步骤中经历大的构象变化。这个家族的成员包括肌动蛋白、己糖激酶和热休克蛋白hsc70。
我们在此报告GK及其与甘油和ADP复合的突变体（Ala65→Thr）的晶体结构。两种酶的晶体都含有相同的222对称四聚体。功能性二聚体与先前描述的IIAGlc-GK复合结构相同。然而，四聚体界面有显著差异，一个功能性二聚体有相对22.3度的旋转和6.34埃的平移。除了两个区域外，整体单体结构没有变化：IIAGlc结合位点发生结构重排，并且残基230 - 236变得有序并在四聚体界面结合正磷酸盐。我们还报告了GK的第二个突变体（Ile474→Asp）与IIAGlc复合的结构；该复合物与野生型IIAGlc-GK复合物同晶型结晶。在IIAGlc结合位点有取代的GK定点突变体显示出显著改变的动力学和调节特性，表明结合位点的构象与活性调节相关。
我们得出结论，这里呈现的新四聚体结构是生理相关四聚体的非活性形式。正磷酸盐结合位点的结构和位置与它作为FBP结合位点的一部分是一致的。突变分析和IIAGlc-GK（Ile474→Asp）复合物的结构表明IIAGlc结合位点的构象转变是IIAGlc调节的一个重要方面。
