title: Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase.
abstract: 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).
authors.affiliation: Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig.
authors.forename: Antje
authors.initials: A
authors.lastname: Brüser
authors.affiliation: Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Kirchberger
authors.affiliation: Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Kloos
authors.affiliation: Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Sträter
authors.affiliation: Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig. Electronic address: schoberg@medizin.uni-leipzig.de.
authors.forename: Torsten
authors.initials: T
authors.lastname: Schöneberg
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation / Q000502:physiology; D001665:Binding Sites; D019143:Evolution, Molecular; D006801:Humans; D009154:Mutation; D025203:Phosphofructokinase-1, Muscle Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D025203:Phosphofructokinase-1, Muscle Type; C026043:PFKM protein, human