Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig.
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
J Biol Chem. 2012 May 18;287(21):17546-17553. doi: 10.1074/jbc.M112.347153. Epub 2012 Apr 3.
6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).
6-磷酸果糖激酶（Pfk）是同型和异型寡聚体，变构酶，催化糖酵解的限速步骤之一：在位置 1上磷酸化果糖 6-磷酸。Pfk 活性受多种调节剂的调节，包括腺嘌呤核苷酸。最近来自真核 Pfk 的晶体结构揭示了几个腺嘌呤核苷酸结合位点。在此，我们通过定点诱变和酶动力学研究确定了两个腺嘌呤核苷酸结合位点的功能相关性。对 Pfk 突变体的后续表征允许鉴定激活（AMP、ADP）和抑制（ATP、ADP）变构结合位点。一个结合位点的突变通过与另一个结合位点相互作用的核苷酸反过来影响变构调节。这种激活和抑制结合位点之间的相互关联与变构酶调节的现有模型一致。由于真核 Pfk 中的变构核苷酸结合位点不是从原核祖先进化而来的，因此功能相反的变构结合位点之间的相互关联必须在原核和真核 Pfk 中独立发展（趋同进化）。
