Mutagenesis of the fructose‐6‐phosphate−binding site in the 2‐kinase domain of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase

• Published in: European Journal of Biochemistry 1998-06, Vol.254 (3), p.490-496
• Main Authors: Bertrand, Luc, Vertommen, Didier, Freeman, Phillip M., Wouters, Johan, Depiereux, Eric, Di Pietro, Attilio, Hue, Louis, Rider, Mark H.
• Format: Article
• Language: English
• Subjects: 6‐phosphofructo‐2‐kinase; adenylate kinase; Base Sequence; Binding Sites; Biochemistry, Molecular Biology; DNA Primers; fructose 6‐phosphate; Fructosephosphates - metabolism; glycolysis; Kinetics; Life Sciences; Multienzyme Complexes - genetics; Multienzyme Complexes - isolation & purification; Multienzyme Complexes - metabolism; mutagenesis; Mutagenesis, Site-Directed; Phosphofructokinase-2; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - isolation & purification; Phosphoric Monoester Hydrolases - metabolism; Phosphotransferases - genetics; Phosphotransferases - isolation & purification; Phosphotransferases - metabolism; Plasmids; Protein Conformation; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity
• ISSN: 0014-2956; 1432-1033; 1432-1327
• DOI: 10.1046/j.1432-1327.1998.2540490.x

Multiple alignment of several isozyme sequences of the bifunctional enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase revealed conserved residues in the 2‐kinase domain. Among these residues, three asparagine residues (Asn76, Asn97 and Asn133; numbering refers to the liver isozyme sequence) and three threonine residues (Thr132, Thr134 and Thr135) are located near the fructose 6‐phosphate−binding site in the crystal structure of the bifunctional enzyme. The role of these residues in substrate binding and catalysis in the 6‐phosphofructo‐2‐kinase domain has been studied by mutagenesis to alanine. Since the crystal structure of 6‐phosphofructo‐2‐kinase does not contain fructose 6‐phosphate, this substrate was docked into the putative binding site by computer modelling, and its interactions with the protein were predicted. Analysis of the mutagenesis‐induced changes in kinetic properties and of the substrate‐docking model revealed that all these residues are directly or indirectly involved in fructose‐6‐phosphate binding. All the mutants displayed an increased Km for fructose 6‐phosphate (10−200‐fold). We propose that Asn133 stabilises Arg138, which itself makes a direct electrostatic bond with the 6‐phosphate group of fructose 6‐phosphate, that Asn76 interacts with the C3 hydroxyl group of fructose 6‐phosphate, that Thr132 makes a hydrogen bond with the C6 oxygen of this substrate, and that Thr134 interacts with two residues involved in fructose‐6‐phosphate binding, Thr132 and Tyr199. On the other hand, Asn97 and Thr135 play structural roles, by maintaining the structure of the fructose‐6‐phosphate‐binding pocket.