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 (GX4GK(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.
Vertommen
D
D
Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology and the University of Louvain Medical School, Brussels, Belgium.
Bertrand
L
L
Sontag
B
B
Di Pietro
A
A
Louckx
M P
MP
Vidal
H
H
Hue
L
L
Rider
M H
MH
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000378:metabolism
D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D013912:Threonine; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D008239:Lysine