The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase - Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX(4)GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif, Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding, We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site directed mutagenesis, Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine, Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type, The mutations had no effect on fructose-2,6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3',5'-AMP-dependent protein kinase, Binding experiments with ATP, ADP, and their analogs (3'-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role, Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.