代谢重编程，包括葡萄糖摄取增加和乳酸排泄，是癌症的标志。引导葡萄糖降解的糖酵解“门卫”酶磷酸果糖激酶-1（PFK1）在癌症中发生异常调节。虽然已经证明了肿瘤中PFK1活性和表达的改变，但对癌相关体细胞突变的影响知之甚少。PFK1的体细胞突变通过确定参与酶活性调节的关键氨基酸残基，有助于我们了解调度质的调节。本文中，我们描述了人血小板（PFKP）和肝脏（PFKL）亚型中破坏一个在进化上保守的天冬酸和精氨酸之间的盐桥的突变。通过使用纯化重组蛋白，我们展示了在两个PFK1亚型中破坏Asp-Arg对会降低酶活性并改变调度质调节。我们解析了PFK1的晶体结构，分辨率为3.6Å，利用分子动力学模拟来理解改变的调度质调节的分子机制。我们展示了PFKP-D564N具有较低的总体系统能量，并且在效应位点的静电表面电位上发生改变。表达PFKP-D564N的细胞显示了较低的酵解作用速率，而在低细胞能量条件下引发酵解通量的能力与表达野生型PFKP的细胞相比有所增强。总的来说，这些结果表明在催化-调度质结构域界面上的天冬酸-精氨酸突变稳定了t态，并为癌症治疗开发提供了新的机制洞察。2023版权所有：作者。

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic "gatekeeper" enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared to cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.Copyright 2023 The Author(s).