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.

中文翻译：

---

三种人类磷酸果糖激酶之间的生化和转录水平差异表明每种异构体针对特定代谢生态位进行了优化


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)。脊椎动物具有三种 PFK 同工型（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 152 µM；K0.5F6P 147 µM），PFK-P 的亲和力最低（K0.5ATP 276 µM；K0.5F6P 1333 µM），并且PFK-L 表现出高 ATP 亲和力和低 F6P 亲和力的混合图（K0.5ATP 160 µM；K0.5F6P 1360 µM）。与 PFK-L 和 PFK-P 相比，PFK-M 对 ATP 抑制具有更强的抵抗力（特异性常数分别降低 23%、31%、50%）。 GTP 是替代磷酸供体。接口 2 调节非活性二聚体到活性四聚体的平衡，不同异构体之间存在差异，导致四聚体稳定性不同。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] 1.10、0.92、0.54，分别）。两个变构位点的结构分析表明，一个可能专门针对 AMP/ADP，另一个则专门针对较小/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，同时保留调节能力。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 产生）转录水平的同时增加，表明癌症中代谢灵活性的优先考虑。 我们的结果描述了三种 PFK 同工型之间的动力学和转录水平差异，解释了如何针对不同的角色优化每种同工型。