Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.
J Biol Chem. 2022 Jan;298(1):101520. doi: 10.1016/j.jbc.2021.101520. Epub 2021 Dec 21.
Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders.
葡萄糖-6-磷酸酶催化亚基 1（G6PC1）在禁食期间通过介导糖异生和糖原分解途径的末端步骤在肝脏葡萄糖产生中发挥关键作用。与辅助转运蛋白一起，这种膜整合酶催化葡萄糖-6-磷酸（G6P）产生葡萄糖，以支持血糖稳态。与代谢功能一致，G6PC1 基因表达的失调导致糖尿病，并且削弱磷酸水解酶活性的突变构成糖原贮积病 1a 型的临床基础。尽管与健康和疾病相关，但由于缺乏分离具有功能形式的酶的表达和纯化策略，因此对 G6PC1 结构和机制的全面了解受到限制。在本报告中，我们应用了一系列生物物理和生化工具来指纹鉴定以 lauryl maltose neopentyl glycol（LMNG）去污剂胶束形式可溶的催化活性 G6PC1 的体外属性。从 Sf9 昆虫细胞膜中纯化时，糖基化的鼠同源物（mG6PC1）再现了以前在完整肝微粒体中观察到的功能特性，并显示出迄今为止报道的最高比活性。此外，我们的结果建立了 mG6PC1 的催化和结构稳定性之间的直接相关性，这是由磷脂酰胆碱和胆固醇类似物胆甾醇半琥珀酸赋予的增强的热稳定性所强调的。相比之下，阻止 N-连接糖基化的 N96A 变体降低了热稳定性。本文所述的方法克服了该领域的长期障碍，并为 G6PC1 的机制结构生物学及其在复杂代谢紊乱中的作用的详细分析奠定了必要的基础。
