Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.
Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.
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三种人磷酸果糖激酶之间的生化和转录水平差异显示了针对特定代谢小生境的每种亚型的优化。
DOI：
10.1042/bcj20200656
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发表时间：
2020-11-27
期刊：
影响因子：
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通讯作者：
Walkinshaw MD
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文献类型：
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作者：
Fernandes PM;Kinkead J;McNae I;Michels PAM;Walkinshaw MD
6-磷酸果糖激酶-1-激酶（PFK）四聚体催化果糖6-磷酸（F6P）磷酸化为果糖1,6-二磷酸（F16BP）。脊椎动物有三种PFK亚型（PFK- m、PFK- l和PFK- p）。这项研究首次比较了重组人PFK亚型的动力学、结构和转录水平。在测试条件下，PFK-M对F6P和ATP的亲和力最高（K0.5ATP 152µM; K0.5F6P 147µM）， PFK-P的亲和力最低（K0.5ATP 276µM; K0.5F6P 1333µM）， PFK-L表现出高ATP亲和力和低F6P亲和力的混合情况（K0.5ATP 160µM; K0.5F6P 1360µM）。与PFK-L和PFK-P相比，PFK-M对ATP抑制的抵抗力更强（特异性常数分别降低23%、31%和50%）。GTP是另一种磷酸供体。界面2调节非活性二聚体到活性四聚体的平衡，不同的同工异构体不同，导致不同的四聚体稳定性。在测试条件下，PFK-M对果糖2,6-二磷酸（F26BP）变构调节的敏感性低于PFK-L或PFK-P（变构常数[K0.5ATP+F26BP/K0.5ATP]分别为1.10、0.92和0.54）。两个变构位点的结构分析表明，一个可能专门用于AMP/ADP，另一个可能专门用于较小/灵活的调节剂（柠檬酸盐或磷酸烯醇丙酮酸盐）。PFK-L和PFK-P转录物水平之间的相关性表明，同时表达可能会扩大F16BP产生的代谢能力，同时保持调控能力。对癌症样本的分析揭示了PFK-P和PKM2（丙酮酸激酶M2）之间有趣的相似之处，以及PFK-P和PFKFB3（负责F26BP的产生）转录物水平的同时增加，表明癌症中代谢灵活性的优先级。我们的研究结果描述了三种PFK异构体之间的动力学和转录水平差异，解释了每种异构体如何针对不同的作用进行优化。
6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.