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Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism
Biochemistry ( IF 2.9 ) Pub Date : 2018-02-09 00:00:00 , DOI: 10.1021/acs.biochem.8b00022 Shawn M. Sternisha 1 , Peilu Liu 1 , Alan G. Marshall 1, 2 , Brian G. Miller 1
Biochemistry ( IF 2.9 ) Pub Date : 2018-02-09 00:00:00 , DOI: 10.1021/acs.biochem.8b00022 Shawn M. Sternisha 1 , Peilu Liu 1 , Alan G. Marshall 1, 2 , Brian G. Miller 1
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Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce 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 the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value 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.
中文翻译：
与先天性高胰岛素血症相关的人葡糖激酶的变体中的酶激活的机制起源。
人葡萄糖激酶（GCK）充当人体的主要葡萄糖传感器，并在葡萄糖体内平衡维持中发挥关键作用。gck的功能获得性突变会产生导致先天性高胰岛素血症的过度活跃的酶变异。先前的生物化学和生物物理研究表明，活化的疾病变体可以分为两种机械上不同的类别，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k cat值受到部分扩散限制，而k catβ型变量的值与粘度无关。瞬态化学猝灭流分析表明，野生型GCK和α型变异体表现出爆发动力学，而β型变异体缺乏爆发相。未配体酶的氢-氘交换质谱比较表明，无序的活性位点环（在结合葡萄糖后会折叠）受到保护，无法在α型变体中进行交换。α型变体还显示出位于酶铰链区附近的β链内交换水平的提高，这在葡萄糖结合时会更多地暴露于溶剂中。相比之下，相对于未配体的野生型GCK，β型活化不会引起整体或局部交换的实质性差异。一起，这些结果表明，α型激活是由于未配体的GCK的构象集合向类似于葡萄糖结合构象的状态转变而引起的，而β型激活则归因于产物释放速率的加快。这项工作阐明了天然存在的，活化的GCK疾病变体的分子基础，并提供了对GCK独特的动力学协同性的结构和动态起源的洞察力。
更新日期：2018-02-09
中文翻译：
与先天性高胰岛素血症相关的人葡糖激酶的变体中的酶激活的机制起源。
人葡萄糖激酶（GCK）充当人体的主要葡萄糖传感器，并在葡萄糖体内平衡维持中发挥关键作用。gck的功能获得性突变会产生导致先天性高胰岛素血症的过度活跃的酶变异。先前的生物化学和生物物理研究表明，活化的疾病变体可以分为两种机械上不同的类别，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k cat值受到部分扩散限制，而k catβ型变量的值与粘度无关。瞬态化学猝灭流分析表明，野生型GCK和α型变异体表现出爆发动力学，而β型变异体缺乏爆发相。未配体酶的氢-氘交换质谱比较表明，无序的活性位点环（在结合葡萄糖后会折叠）受到保护，无法在α型变体中进行交换。α型变体还显示出位于酶铰链区附近的β链内交换水平的提高，这在葡萄糖结合时会更多地暴露于溶剂中。相比之下，相对于未配体的野生型GCK，β型活化不会引起整体或局部交换的实质性差异。一起，这些结果表明，α型激活是由于未配体的GCK的构象集合向类似于葡萄糖结合构象的状态转变而引起的，而β型激活则归因于产物释放速率的加快。这项工作阐明了天然存在的，活化的GCK疾病变体的分子基础，并提供了对GCK独特的动力学协同性的结构和动态起源的洞察力。
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