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 ANDTHR-55 BY SITE-DIRECTED MUTAGENESIS/

All known phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX(4)GK(S/T)) in the 6-phosphofructo-2-kinase doma in corresponding to the so-called nucleotide binding fold signature or Walker A motif, Mutagenesis and crystal structure data from several n ucleotide binding proteins, which also contain this sequence, showed t he importance of the lysine and serine/threonine residues in nucleotid e binding, We have studied the role of Lys-54 and Thr-55 in MgATP bind ing in the 6-phosphofructo-2-kinase domain of rat liver phosphofructo- 2-kinase/fructose-2,6-bisphosphatase by site directed mutagenesis, Lys -54 was mutated to methionine, whereas Thr-55 was mutated to valine, s erine, 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-ki nase activity compared with the wild type, The mutations had no effect on fructose-2,6-bisphosphatase activity and did not affect the activa tion of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3' ,5'-AMP-dependent protein kinase, Binding experiments with ATP, ADP, a nd their analogs (3'-N-methylanthraniloyl derivatives) showed that the se two residues do not play the same role, Lys-54 is involved in ATP b inding, whereas Thr-55 is important for catalysis.