The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.
To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.
Cárcamo
J G
JG
Instituto de Bioquímica, Universidad Austral de Chile, Valdivia, Chile.
Yañez
A J
AJ
Ludwig
H C
HC
León
O
O
Pinto
R O
RO
Reyes
A M
AM
Slebe
J C
JC
D000249:Adenosine Monophosphate / Q000494:pharmacology*; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D004926:Escherichia coli; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005635:Fructosediphosphates / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013696:Temperature
D004791:Enzyme Inhibitors; D005635:Fructosediphosphates; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium