Binding Kinetics of Glucose and Allosteric Activators to Human Glucokinase Reveal Multiple Conformational States
Binding Kinetics of Glucose and Allosteric Activators to Human Glucokinase Reveal Multiple Conformational States
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DOI：
10.1021/bi900374c
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发表时间：
2009-06-16
期刊：
影响因子：
2.9
通讯作者：
Ferry, Gilles
中科院分区：
文献类型：
--
作者：
Antoine, Mathias;Boutin, Jean A.;Ferry, Gilles
缓慢的构象变化被认为是葡萄糖激酶（GK）与葡萄糖的动力学正协同效应的原因。诱导拟合和预先存在的平衡动力学模型已经提出。在本研究中，平衡和前稳态荧光光谱已被用来解决这些相互矛盾的报告。在apo-GK与葡萄糖快速混合后观察到多相瞬变。进展曲线分析显示与诱导拟合模型不一致。主要动力学阶段的葡萄糖依赖性支持至少两种缓慢相互转化的GK构象异构体的存在。在不存在葡萄糖的情况下，约80%的GK群体可能处于大部分开放构象（K-D约为30 mM）。剩余的20%处于更紧凑的构象（K-D约为0.2mM）。瞬态显示三个额外的阶段可能反映中间体之间的超开放和封闭构象的途径。使用固有荧光GK激活剂（GKA），表明GK可以在不存在葡萄糖的情况下结合GKA，证实了预先存在的平衡模型的有效性。此外，一个扰动的GKA结合动力学参数后，预平衡封闭GK与ATP类似物建议的变构位点后，核苷酸结合的局部重排。我们的数据表明，在没有任何配体的情况下，GK可能能够广泛地采样的构象空间划定的超开和封闭的构象。当配体如葡萄糖或GKA与特定GK构象异构体结合时，复合物GK构象平衡容易发生变化。
Slow conformational changes have been proposed to be responsible for the kinetic positive cooperativity of glucokinase (GK) with glucose. Induced-fit and preexisting equilibrium kinetic models have been previously suggested. In the present study, equilibrium and pre-steady-state fluorescence spectroscopy has been used to resolve those conflicting reports. Multiphasic transients were observed after rapid mixing of apo-GK with glucose. Progress curve analysis revealed inconsistencies with the induced-fit model. The glucose dependence of the major kinetic phase supported the preexistence of at least two slowly interconverting GK conformers. In the absence of glucose, approximate to 80% of the GK population is likely poised in a largely open conformation (K-D approximate to 30 mM). The remaining 20% is in a more compact conformation (K-D approximate to 0.2 mM). Transients revealed three additional phases likely reflecting intermediates on the pathway between the superopen and the closed conformer. Using an intrinsically fluorescent GK activator (GKA), it was shown that GK can bind GKA in the absence of glucose, confirming the validity of the preexisting equilibrium model. Additionally, a perturbation of the GKA binding kinetic parameters after preequilibration of closed GK with an ATP analogue suggested a local rearrangement of the allosteric site upon nucleotide binding. Our data suggest that, in the absence of any ligand, GK might be able to extensively sample the conformational space delimited by the superopen and the closed conformations. The complex GK conformational equilibrium is readily shifted upon binding of ligands such as glucose or GKAs on specific GK conformers.