PMID:9032446

Citation	Bertrand, L, Deprez, J, Vertommen, D, Di Pietro, A, Hue, L and Rider, MH (1997) Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem. J. 321 ( Pt 3):623-7
Abstract	In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.
Links	PubMed PMC1218115
Keywords	Adenosine Triphosphate/metabolism; Animals; Circular Dichroism; Cloning, Molecular; Enzyme Stability/drug effects; Escherichia coli/genetics; Fructose-Bisphosphatase/genetics; Fructose-Bisphosphatase/metabolism; Gene Expression/genetics; Guanidine; Guanidines/pharmacology; Hydrogen-Ion Concentration; Kinetics; Liver/enzymology; Models, Chemical; Mutagenesis, Site-Directed/genetics; Mutation; Phosphofructokinase-1/genetics; Phosphofructokinase-1/metabolism; Phosphofructokinase-2; Phosphotransferases (Alcohol Group Acceptor)/chemistry; Phosphotransferases (Alcohol Group Acceptor)/genetics; Protein Binding/genetics; Protein Denaturation; Rats; Recombinant Proteins/genetics; Recombinant Proteins/metabolism
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