Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions.
Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions.
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DOI：
10.1099/00221287-147-2-391
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发表时间：
2001-02
期刊：
影响因子：
1.5
通讯作者：
Amanda K. Pearce;Kay Crimmins;E. Groussac;Michael J. E. Hewlins;J. Richard Dickinson;Jean Francois;Ian R. Booth;Alistair J. P. Brown
中科院分区：
文献类型：
--
作者：
Amanda K. Pearce;Kay Crimmins;E. Groussac;Michael J. E. Hewlins;J. Richard Dickinson;Jean Francois;Ian R. Booth;Alistair J. P. Brown
酵母磷酸果糖-1-激酶（Pf 1 k）和丙酮酸激酶（Pyk 1）是变构调节的酶，催化糖酵解中基本上不可逆的反应。这些酶的合成和活性都受到严格的调节。为了将Pf 1 k和Pyk 1合成的控制与它们的变构调节实验分离，构建了PFK 1、PFK 2和PYK 1突变体的同源组，其中这些野生型编码区由替代启动子驱动。携带PGK 1启动子融合的突变体显示正常的生长速率、葡萄糖消耗和乙醇生产，表明Pyk 1和Pf 1 k合成的相对严格的调节对于发酵生长条件下的糖酵解控制不是必需的。携带与PGK 1启动子的增强子较少版本（PGK 1（Delta 767））融合的突变体表达的Pyk 1和Pf 1 k水平比正常低约2.5倍。PFK 1和PFK 2双突变体的生理和代谢分析表明，减少Pf 1 k对生长没有显着影响，显然是由于其正效应，果糖2，6-二磷酸的补偿性增加。相比之下，生长速率和糖酵解通量在PGK 1（Delta 767）-PYK 1突变体中降低，该突变体具有降低的Pyk 1水平。出乎意料的是，Pyk 1水平的降低导致TCA循环的碳流量增加，即使在发酵生长条件下也是如此。因此，Pyk 1对酵母中碳通量的速率和方向都具有显著的控制水平。
Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated. To separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of PFK1, PFK2 and PYK1 mutants was constructed in which these wild-type coding regions were driven by alternative promoters. Mutants carrying PGK1 promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions. Mutants carrying fusions to an enhancer-less version of the PGK1 promoter (PGK1(Delta767)) expressed Pyk1 and Pf1k at about 2.5-fold lower levels than normal. Physiological and metabolic analysis of the PFK1 PFK2 double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the PGK1(Delta767)-PYK1 mutant, which had decreased Pyk1 levels. Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast.