Pearce Amanda K, Crimmins Kay, Groussac Evelyne, Hewlins Michael J E, Dickinson J Richard, Francois Jean, Booth Ian R, Brown Alistair J P
Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK1.
Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504 UR-INRA 792, Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, 31077 Toulouse Cedex 04, France2.
Microbiology (Reading). 2001 Feb;147(Pt 2):391-401. doi: 10.1099/00221287-147-2-391.
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.
酵母磷酸果糖激酶-1（Pf1k）和丙酮酸激酶（Pyk1）是变构调节酶，它们催化糖酵解中基本不可逆的反应。这些酶的合成和活性都受到严格调控。为了通过实验将Pf1k和Pyk1合成的调控与其变构调节分开，构建了一组同基因的PFK1、PFK2和PYK1突变体，其中这些野生型编码区由替代启动子驱动。携带PGK1启动子融合的突变体显示出正常的生长速率、葡萄糖消耗和乙醇产量，这表明在发酵生长条件下，对Pyk1和Pf1k合成的相对严格调控对于糖酵解控制并非必不可少。携带与无增强子版本的PGK1启动子（PGK1(Delta767)）融合的突变体表达的Pyk1和Pf1k水平比正常水平低约2.5倍。PFK1 PFK2双突变体的生理和代谢分析表明，Pf1k水平降低对生长没有显著影响，这显然是由于其正效应物果糖2,6-二磷酸的补偿性增加。相比之下，PGK1(Delta767)-PYK1突变体的生长速率和糖酵解通量降低，该突变体的Pyk1水平降低。出乎意料的是，即使在发酵生长条件下，Pyk1水平降低也导致碳流向三羧酸循环增加。因此，Pyk1对酵母中碳通量的速率和方向都发挥着显著的控制作用。
