Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates
Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates
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DOI：
10.1128/ec.2.3.599-608.2003
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发表时间：
2003-06-01
期刊：
影响因子：
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通讯作者：
Nielsen, J
中科院分区：
文献类型：
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作者：
dos Santos, MM;Gombert, AK;Nielsen, J
酿酒酵母（Saccharomyces cerevisiae）中心代谢网络的详细描述。PK 113-7D在共代谢过程中进行。葡萄糖和乙酸的不同混合物，使用有氧限制c的趋化剂，其中这两种底物中的一种被标记为C-13。为了证实苹果酸酶的作用，我们在相同的条件下培养了一个删除了相应基因的等基因菌株。分析了蛋白质氨基酸的标记模式，并用于估计代谢通量和/或推断中心碳代谢和氨基酸生物合成酶的体内活性。苹果酶通量随乙酸含量的增加呈线性增加。在高乙酸部分的生长过程中，苹果酸酶的活性满足了线粒体中丙酮酸的生物合成需要，而在细胞质中丙酮酸则通过丙酮酸激酶提供。在一些情况下，酶活性出乎意料地被检测到，例如，乙酸极低的部分的乙醛酸分流，磷酸烯醇丙酮酸羧激酶的乙酸部分为0.46 C-mol的乙酸/C-mol的底物，葡萄糖通过三羧酸循环分解为CO2的乙酸非常高的部分。细胞质中丙氨酸转氨酶活性测定，发现α -异丙基苹果酸合成酶在体内有两种活性形式，一种是线粒体形式，另一种是短细胞质形式。
A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism. of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with C-13. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO2 via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that alpha-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.