Abstract

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 AMPbinding 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 [Siebe 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 1,6bisphosphatase 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 Lysl 12 appears to be the reactive residue involved in the AMP sensitivity. According to the fructose 1,6-bisphosphatase crystal structure, LysSO is strategically positioned at the C1-C2 interface, near the molecular center of the tetramer, and Lysl 12 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. © 1999 Plenum Publishing Corporation.