Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism.
Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism.
复制标题
与先天性高胰岛素血症相关的人类葡萄糖激酶变体中酶激活的机制起源。
DOI：
10.1021/acs.biochem.8b00022
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Miller,BrianG
中科院分区：
文献类型：
--
作者：
Sternisha,ShawnM;Liu,Peilu;Marshall,AlanG;Miller,BrianG
人葡萄糖激酶（GCK）作为人体的主要葡萄糖传感器，在维持葡萄糖稳态中起着关键作用。功能获得性突变产生过度活跃的酶变体，导致先天性高胰岛素血症。先前的生物化学和生物物理学研究表明，活化的疾病变体可以分为两种机制上不同的类别，称为α型和β型。稳态粘度变化研究表明，野生型GCK和α型变体的kcat值是部分扩散限制的，而β型变体的kcat值是粘度无关的。瞬态化学猝灭流分析表明，野生型GCK和α-型变体显示爆发动力学，而β-型变体缺乏爆发相。未配体酶的比较氢-氘交换质谱表明，在α型变体中，一个无序的活性位点环（在与葡萄糖结合时折叠）受到保护，不发生交换。α型变体还显示出位于酶铰链区附近的β链内的交换水平增加，其在葡萄糖结合后变得更多地暴露于溶剂。相比之下，β-型激活导致相对于未配体的野生型GCK的全局或局部交换没有实质性差异。总之，这些结果表明，α型活化是由于未配体GCK的构象整体向类似于葡萄糖结合构象的状态转变所致，而β型活化可归因于产物释放速率加快。这项工作阐明了自然发生的，激活GCK疾病变异的分子基础，并提供了深入了解GCK独特的动力学协同性的结构和动力学起源。
Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations ingckproduce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that thekcatvalues of wild-type GCK and an α-type variant are partially diffusion-limited, whereas thekcatvalue of a β-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the β-type variant lacks a burst phase. Comparative hydrogen–deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active site loop, which folds upon binding of glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a β-strand located near the enzyme’s hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, β-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas β-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK’s unique kinetic cooperativity.