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.
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.
Bertrand
L
L
University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Deprez
J
J
Vertommen
D
D
Di Pietro
A
A
Hue
L
L
Rider
M H
MH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D025481:Phosphofructokinase-2; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D006146:Guanidines; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010732:Phosphofructokinase-1; D006597:Fructose-Bisphosphatase; D019791:Guanidine