The Effects of Glucokinase Polymerization During Metabolic Transitions

 描述（由申请人提供）：该项目的主要目标是了解代谢网络如何响应环境营养素浓度的突然变化而进行调节。虽然我们对各种稳定状态下的中心碳代谢有了很好的了解，但尚不清楚这些代谢网络如何适应在太短而无法改变蛋白质组组成的时间尺度上保持活力。我们专注于糖酵解的第一步是如何调节的，当细胞在乙醇上缓慢生长时，暴露于足够高的葡萄糖浓度，通过糖酵解诱导高通量，支持快速发酵生长。开始糖酵解太快会耗尽ATP并杀死细胞。初步研究表明，代谢酶的聚合在调节这种转变的动力学中起着至关重要的作用。我们已经发现，葡萄糖激酶-1（Glk 1），负责催化糖酵解第一步的三种酶之一，在呼吸细胞时形成线性聚合物 遇到高葡萄糖浓度。纯化的Glk 1在葡萄糖和ATP存在下形成聚合物，聚合抑制Glk 1的催化活性。利用遗传学，我们已经证明，删除GLK 1减少了细胞死亡，但减缓了呼吸细胞遇到葡萄糖时生长速度的加速。利用所有生命王国中糖酵解的保守性，我们将研究饥饿酿酒酵母的代谢对突然引入富含葡萄糖的培养基的反应方式，作为对环境葡萄糖浓度快速增加的一般反应的易处理模型。我们将在多个尺度上解决这个问题：使用遗传学，生物化学和显微镜的组合来了解Glk 1及其聚合如何影响糖酵解和细胞生长;使用生物化学和结构生物学的组合来阐明Glk 1聚合如何降低Glk 1酶活性;并确定这些机制的保守性。

 DESCRIPTION (provided by applicant): The major goal of this project is to understand how metabolic networks are regulated in response to sudden changes in the concentration of environmental nutrients. While we have a good understanding of central carbon metabolism in various steady states, it is unclear how these metabolic networks adapt to maintain viability on timescales that are too short to alter the composition of the proteome. We focus on how the first step in glycolysis is regulated when cells growing slowly on ethanol are exposed to high enough glucose concentrations to induce the high flux through glycolysis that supports rapid, fermentative growth. Starting glycolysis too fast can deplete ATP and kill cells. Preliminary studies suggest that polymerization of a metabolic enzyme plays a crucial role in regulating the kinetics of this transition. We have found that Glucokinase-1 (Glk1), one of the three enzymes responsible for catalyzing the first step in glycolysis, forms linear polymers when respiring cells encounter high glucose concentrations. Purified Glk1 forms polymers in the presence of glucose and ATP and polymerization inhibits Glk1's catalytic activity. Using genetics, we have demonstrated that deleting GLK1 reduces cell death but slows the acceleration of growth rate when respiring cells encounter glucose. Taking advantage of the conservation of glycolysis across all kingdoms of life, we will investigate the way that starved Saccharomyces cerevisiae's metabolism responds to sudden introduction into glucose-rich media as a tractable model for the general response to rapid increases in the environmental glucose concentration. We will tackle this problem at multiple scales: using a combination of genetics, biochemistry and microscopy to understand how Glk1 and its polymerization affect both glycolysis and cellular growth; using a combination of biochemistry and structural biology in order to elucidate how Glk1 polymerization reduces Glk1 enzymatic activity; and determining the conservation of these mechanisms.