Mutagenesis of the fructose-6-phosphate-binding site in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
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.
Bertrand
L
L
Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Vertommen
D
D
Freeman
P M
PM
Wouters
J
J
Depiereux
E
E
Di Pietro
A
A
Hue
L
L
Rider
M H
MH
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005636:Fructosephosphates / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D005636:Fructosephosphates; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C027618:fructose-6-phosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases