Revisiting the 13C-label distribution of the non-oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence
Revisiting the 13C-label distribution of the non-oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence
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DOI：
10.1111/j.1742-4658.2005.04907.x
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发表时间：
2005-10-01
期刊：
影响因子：
5.4
通讯作者：
Heijnen, JJ
中科院分区：
文献类型：
--
作者：
Kleijn, RJ;van Winden, WA;Heijnen, JJ
目前应用于戊糖磷酸途径的非氧化分支的化学计量通量平衡模型中的反应结构不符合转酮醇酶（EC 2.2.1.1）和转醛醇酶（EC 2.2.1.2）的已建立的乒乓动力学机制。基于乒乓机制，戊糖磷酸途径的非氧化分支的传统反应被代谢物特异性的、可逆的、乙醇醛部分（C-2）和二羟基丙酮部分（C-3）片段产生和消耗半反应所取代。结果表明，化学计量模型的基础上，这些半反应是从根本上不同于目前应用的化学计量模型的独立的C-2和C-3片段池在戊糖磷酸途径的数量，并可能导致不同的标签分布的C-13示踪剂实验。为了研究新的反应结构对细胞内估计通量模式的实际影响，使用了来自先前发表的基于C-13的酿酒酵母代谢通量分析的质量同位素异构体测量。从遗传学的角度来看，包括大肠杆菌和S。酿酒酵母含有编码转酮醇酶和转醛醇酶同工酶的多个基因。然而，这些基因产物也被积极表达的程度仍然未知。结果表明，新提出的化学计量模型允许研究同工酶的C-13-标签分布在非氧化分支的戊糖磷酸途径的影响，通过扩展半反应为基础的化学计量模型与两个不同的转酮醇酶，而不是一个。结果表明，列入同工酶影响随后的通量估计。
The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping-pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping-pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C-2) and dihydroxyacetone moiety (C-3) fragments producing and consuming half-reactions. It is shown that a stoichiometric model based upon these half-reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C-2 and C-3 fragment pools in the pentose phosphate pathway and can lead to different label distributions for C-13-tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published C-13-based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several microorganisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the C-13-label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the half-reaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.