Order-disorder transitions govern kinetic cooperativity and allostery of monomeric human glucokinase.
Order-disorder transitions govern kinetic cooperativity and allostery of monomeric human glucokinase.
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秩序转变控制了单体人葡萄糖酶的动力学合作和变构。
DOI：
10.1371/journal.pbio.1001452
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发表时间：
2012
期刊：
影响因子：
9.8
通讯作者：
Brüschweiler R
中科院分区：
文献类型：
--
作者：
Larion M;Salinas RK;Bruschweiler-Li L;Miller BG;Brüschweiler R
人类葡萄糖激酶的功能动力学分析揭示了一个缓慢的有序-无序过渡支配着响应葡萄糖浓度的单体动力学协同性。葡萄糖激酶（GCK）在胰腺中催化葡萄糖分解代谢的限速步骤，在那里它作为身体的主要葡萄糖传感器起作用。GCK功能障碍可导致几种潜在的致命疾病，包括年轻型II型糖尿病（MODY-II）和婴儿期持续性低血糖高胰岛素血症（PHHI）。GCK通过对血糖水平升高表现出s型动力学反应来维持葡萄糖稳态。这种正的协同性是独一无二的，因为这种酶只作为一个单体发挥作用，并且只有一个葡萄糖结合位点。尽管近半个世纪的研究，GCK的同向变构的机制基础仍未得到解决。在这里，我们利用17个同位素标记的异亮氨酸甲基和3个色氨酸侧链作为敏感的核磁共振（NMR）探针，从葡萄糖介导的大规模无序-有序转变的角度解释了GCK的协同性。我们发现无配体GCK的小结构域本质上是无序的，并且样品是一个广泛的构象集合。我们还证明，小分子糖尿病治疗剂和高胰岛素相关的GCK突变具有惊人相似的激活机制，其特征是群体向更狭窄、有序的整体转变，类似于葡萄糖结合构象。我们的研究结果支持一个模型，在这个模型中，GCK在低葡萄糖浓度下产生协同动力学响应，通过使用小结构域的毫秒无序循环作为“时滞环路”，在高葡萄糖浓度下绕过它，提供了一种在生理条件下变变调节人类GCK活性的独特机制。葡萄糖激酶是一种关键的代谢酶，是人体主要的葡萄糖传感器。葡萄糖激酶通过一种独特但鲜为人知的对葡萄糖浓度增加的协同动力学反应来调节胰腺分泌胰岛素的速率。葡萄糖激酶基因突变可导致青少年II型糖尿病（MODY II）、永久性新生儿糖尿病（PNDM）和婴儿期低血糖性高胰岛素血症（HI），这一事实强调了该酶的生理重要性。在这项研究中，我们使用尖端的高分辨率核磁共振方法来了解葡萄糖激酶的动力学特性如何促进葡萄糖稳态。我们还试图了解一类最近发现的小分子药物是如何增强葡萄糖激酶活性的，它们有望成为2型糖尿病的治疗药物。我们的结果表明，葡萄糖激酶样品在没有葡萄糖的情况下具有一系列的构象状态。然而，在葡萄糖或小分子激活剂的存在下，酶群转向更狭窄、结构良好的状态集合。我们的研究结果为葡萄糖激酶合作动力学提供了一个新的模型，该模型依赖于对葡萄糖浓度的缓慢有序-无序转变。这些结果也揭示了葡萄糖激酶激活的普遍机制，这可能为开发新的抗糖尿病药物提供信息。
Analysis of the functional dynamics of human glucokinase reveals that a slow order-disorder transition governs monomeric kinetic cooperativity in response to glucose concentrations. Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity–onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder–order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder–order cycle of the small domain as a “time-delay loop,” which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions. Glucokinase is a key metabolic enzyme that functions as the body's principal glucose sensor. Glucokinase regulates the rate at which insulin is secreted by the pancreas by using a unique but poorly understood cooperative kinetic response to increasing glucose concentrations. The physiological importance of this enzyme is underlined by the fact that mutations in the glucokinase gene lead to maturity-onset diabetes of the young type II (MODY II), permanent neonatal diabetes mellitus (PNDM), and hypoglycemic hyperinsulinemia of infancy (HI). In this study, we use cutting-edge high-resolution nuclear magnetic resonance methods to understand how the kinetic properties of glucokinase contribute to glucose homeostasis. We also seek to understand how a class of recently discovered small-molecule drugs, which hold promise as therapeutics for type 2 diabetes, function to enhance glucokinase activity. Our results suggest that glucokinase samples a range of conformational states in the absence of glucose. However, in the presence of glucose or a small-molecule activator, the enzyme population shifts towards a more narrow, well-structured ensemble of states. Our findings provide a new model for glucokinase cooperative kinetics, which relies on a slow order–disorder transition in response to glucose concentrations. These results also reveal a universal mechanism of glucokinase activation, which may inform the development of new antidiabetic agents.
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