Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways

Glycolysis is the core metabolic
pathway supplying energy to cells.
Whereas the vast majority of studies focus on specific aspects of
the process, global analyses characterizing simultaneously all enzymes
involved in the process are scarce. Here, we demonstrate that quantitative
label- and standard-free proteomics allows accurate determination
of titers of metabolic enzymes and enables simultaneous measurements
of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose
1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence
of tissue-specific isoenzymes bearing different kinetic properties,
the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy
metabolism, we analyzed titers of the enzymes in additional 7 mouse
organs and in human cells. Across the analyzed samples, we identified
two basic profiles: a “fast glucose uptake” one in brain
and heart, and a “gluconeogenic rich” one occurring
in liver. In skeletal muscles and other organs, we found intermediate
profiles. Obtained data highlighted the glucose-flux-limiting role
of hexokinase which activity was always 10- to 100-fold lower than
the average activity of all other glycolytic enzymes. A parallel determination
of enzyme titers and maximal enzymatic activities allowed determination
of kcat values without enzyme purification.
Results of our in-depth proteomic analysis of the mouse organs did
not support the concepts of regulation of glycolysis by lysine acetylation.

糖酵解是向细胞提供能量的核心代谢途径。尽管绝大多数研究集中于该过程的特定方面，但全面分析同时表征该过程所有酶的全球分析却颇为罕见。在本研究中，我们展示了定量标记和无标准蛋白组学技术能够精确测定代谢酶的滴度，并使得对小鼠大脑、肝脏和肌肉中所有糖酵解酶及糖异生途径中的果糖-1,6-二磷酸酶的滴度和最大酶活性（Amax）的同步测量成为可能。尽管存在具有不同动力学特性的组织特异性同工酶，但酶的滴度通常与Amax值具有良好的相关性。为呈现能量代谢的更全面图景，我们对另外7个小鼠器官和人类细胞中的酶滴度进行了分析。在分析样本中，我们识别出两种基本谱型：大脑和心脏中的“快速葡萄糖摄取”型，以及肝脏中的“富含糖异生”型。在骨骼肌和其他器官中，我们发现存在中间型谱型。获得的数据突出了己糖激酶在葡萄糖通量限制中的作用，其活性通常比所有其他糖酵解酶的平均活性低10至100倍。通过平行测定酶滴度和最大酶活性，我们能够在不进行酶纯化的情况下确定kcat值。我们对小鼠器官进行深入蛋白组学分析的结果不支持通过赖氨酸乙酰化调节糖酵解的概念。