Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids
Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids
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DOI：
10.1046/j.1432-1327.2001.02126.x
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发表时间：
2001-04-01
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影响因子：
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通讯作者：
Szyperski, T
中科院分区：
文献类型：
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作者：
Maaheimo, H;Fiaux, J;Szyperski, T
利用蛋白质氨基酸的生物合成部分C - 13标记，在以葡萄糖为唯一碳源的基本培养基中进行分批培养，对酿酒酵母细胞的有氧和无氧中心代谢进行了研究。这首先能够解析细胞质和线粒体中活跃的中心代谢途径网络，其次能够确定表征糖酵解、戊糖磷酸循环、三羧酸循环和C1代谢的通量比，第三能够评估丙酮酸、乙酰辅酶A、草酰乙酸和甘氨酸的区间转运通量。数据还表明丙氨酸氨基转移酶位于线粒体中，并且氨基酸是按照已有文献记载的途径合成的。在有氧和无氧条件下：(a)线粒体甘氨酸裂解途径是活跃的，并且观察到甘氨酸向细胞质的外排；(b)戊糖磷酸途径仅用于生物合成，即磷酸烯醇式丙酮酸完全通过糖酵解产生；(c)大部分细胞质草酰乙酸是通过丙酮酸的回补羧化作用合成的；(d)苹果酸酶对线粒体丙酮酸代谢起关键作用；(e)草酰乙酸从细胞质到线粒体的转运在很大程度上是单向的，苹果酸 - 天冬氨酸穿梭和琥珀酸 - 富马酸载体的活性较低；(e)大部分线粒体丙酮酸是从细胞质输入的；以及(f)乙醛酸循环是不活跃的。在有氧条件下，75%的线粒体草酰乙酸来自丙酮酸的回补羧化作用，而在无氧条件下，三羧酸循环以分支方式运行，仅满足生物合成需求。本研究表明，氨基酸的部分C - 13标记是研究有隔室的真核系统的一种有力方法。
Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional C-13 labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional C-13 labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.