MUTAGENESIS OF THE FRUCTOSE-6-PHOSPHATE-BINDING SITE IN THE 2-KINASE DOMAIN OF 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BIPHOSPHATASE/

Multiple alignment of several isozyme sequences of the bifunctional en zyme phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed conse rved residues in the 2-kinase domain. Among these residues, three aspa ragine residues (Asn76, Asn97 and Asn133; numbering refers to the live r isozyme sequence) and three threonine residues (Thr132, Thr134 and T hr135) are located near the fructose 6-phosphate-binding site in the c rystal structure of the bifunctional enzyme. The role of these residue s in substrate binding and catalysis in the 6-phosphofructo-2-kinase d omain has been studied by mutagenesis to alanine. Since the crystal st ructure of 6-phosphofructo-2-kinase does not contain fructose 6-phosph ate, this substrate was docked into the putative binding site by compu ter modelling, and its interactions with the protein were predicted. A nalysis of the mutagenesis-induced changes in kinetic properties and o f the substrate-docking model revealed that all these residues are dir ectly or indirectly involved in fructose-6-phosphate binding. All the mutants displayed an increased K-m for fructose 6-phosphate (10-200-fo ld). We propose that Asn133 stabilises Arg138, which itself makes a di rect electrostatic bond with the 6-phosphate group of fructose 6-phosp hate, that Asn76 interacts with the C3 hydroxyl group of fructose 6-ph osphate, that Thr132 makes a hydrogen bond with the C6 oxygen of this substrate, and that Thr134 interacts with two residues involved in fru ctose-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.