Suppression of kinetic AMP cooperativity of fructose-1,6-bisphosphatase by carbamoylation of lysine 50.
Selective treatment of pig kidney fructose 1,6-bisphosphatase with cyanate leads to the formation of an active carbamoylated derivative that shows no cooperative interaction between the AMP-binding sites, but completely retains the sensitivity to the inhibitor. By an exhaustive carbamoylation of the enzyme a derivative is formed that has a complete loss of cooperativity and a decrease of sensitivity to AMP. It was proposed that the observed changes of allosteric properties were due to the chemical modification of two lysine residues per enzyme subunit [Slebe et al. (1983), J. Protein Chem. 2, 437-443]. Studies of the temperature dependence of AMP sensitivity and the interaction with Cibacron Blue Sepharose of carbamoylated fructose 1,6-bisphosphatase derivatives indicate that the lysine residue involved in AMP sensitivity is located at the allosteric AMP site, while the lysine residue involved in AMP cooperativity is at a distinct location. Using [14C]cyanate, we identified both lysine residues in the primary structure of the enzyme; Lys50 is essential for AMP cooperativity and Lys112 appears to be the reactive residue involved in the AMP sensitivity. According to the fructose 1,6-bisphosphatase crystal structure, Lys50 is strategically positioned at the C1-C2 interface, near the molecular center of the tetramer, and Lys112 is in the AMP-binding site. The results reported here, combined with the structural data of the enzyme, strongly suggest that the C1-C2 interface is critical for the propagation of the allosteric signal among the AMP sites on different subunits.
Ludwig
H C
HC
Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia.
Herrera
R
R
Reyes
A M
AM
Hubert
E
E
Slebe
J C
JC
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D002846:Chromatography, Affinity; D003485:Cyanates / Q000737:chemistry; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D010449:Peptide Mapping; D013552:Swine
D002250:Carbon Radioisotopes; D003485:Cyanates; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase; D008239:Lysine