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 enzym e phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the anal ogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are l ocated 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 K- m of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000- fold respectively, whereas mutation of Lys-174 to glycine decreased th e V-max 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 mu tations had no effect on the structure and stability of the recombinan t proteins. The results show that Asp-179 and Asp-191 participate in f ructose 6-phosphate binding, whereas Lys-174 is important for catalysi s. Therefore the natural mutation of Lys-174 to glycine in the fbp26 y east isoenzyme could explain the lack of 6-phosphofructo-2-kinase acti vity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.