Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease
Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease
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DOI：
10.1073/pnas.1311990110
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发表时间：
2013-12-17
影响因子：
11.1
通讯作者：
Deane, Janet E.
中科院分区：
文献类型：
--
作者：
Hill, Chris H.;Graham, Stephen C.;Deane, Janet E.
鞘糖脂是哺乳动物细胞膜中普遍存在的成分，溶酶体酶对其分解代谢的缺陷可导致多种疾病。酶β-半乳糖脑苷酶(GALC)的缺乏会导致Krabbe病，这是一种毁灭性的遗传病，其特征是广泛的脱髓鞘和快速、致命的神经变性。在这里，我们介绍了一系列高分辨率的晶体结构，说明了GALC催化循环中的关键步骤。我们已经捕获了短暂的酶-底物复合体的快照，说明了野生型GALC是如何与真正的底物结合的。我们用这种底物对GALC的酶动力学进行了广泛的表征，并通过测定用这种底物长期浸泡晶体后的酶-产物复合体的结构表明，这种酶在晶体中是活性的。我们还确定了共价中间体的结构，该中间体与酶-底物和酶-产物复合体一起，揭示了伴随催化步骤的构象变化，并提供了关键的机理见解，为未来药物伴侣的设计奠定了基础。
Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme beta-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration. Here, we present a series of high-resolution crystal structures that illustrate key steps in the catalytic cycle of GALC. We have captured a snapshot of the short-lived enzyme-substrate complex illustrating how wild-type GALC binds a bona fide substrate. We have extensively characterized the enzyme kinetics of GALC with this substrate and shown that the enzyme is active in crystallo by determining the structure of the enzyme-product complex following extended soaking of the crystals with this same substrate. We have also determined the structure of a covalent intermediate that, together with the enzyme-substrate and enzyme-product complexes, reveals conformational changes accompanying the catalytic steps and provides key mechanistic insights, laying the foundation for future design of pharmacological chaperones.