Suppression of kinetic AMP cooperativity of fructose-1,6-bisphosphatase bycarbamoylation 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 retai ns the sensitivity to the inhibitor. By an exhaustive carbamoylation of the enzyme a derivative is formed that has a complete loss of cooperativity an d a decrease of sensitivity to AMP. It was proposed that the observed chang es of allosteric properties were due to the chemical modification of two ly sine residues per enzyme subunit [Slebe et al. (1983), J. Protein Cihem. 2, 437-443]. Studies of the temperature dependence of AMP sensitivity and the interaction with Cibacron Blue Sepharose of carbamoylated fructose 1,6-bis phosphatase derivatives indicate: that the lysine residue involved in AMP s ensitivity is located at the allosteric AMP site, while the lysine residue involved in AMP cooperativity is at a distinct location. Using [C-14]cyanat e, we identified both lysine residues in the primary structure of the enzym e; Lys50 is essential for AMP cooperativity and Lys112 appears to be the re active residue involved in the AMP sensitivity. According to the fructose 1 ,6-bisphosphatase crystal structure, Lys50 is strategically positioned at t he C1-C2 interface, near the molecular center of the tetramer, and Lys112 i s in the AMP-binding site. The results reported here, combined with the str uctural data of the enzyme, strongly suggest that the C1-C2 interface is cr itical for the propagation of the allosteric signal among the AMP sites on different subunits.