Kinetics and mechanisms of activation and inhibition of porcine liver fructose-1,6-bisphosphatase by monovalent cations.

K+ and Li+ were used to study the kinetic effects of monovalent cations on porcine liver fructose-1,6-bisphosphatase (FBPase). At saturating fructose 1,6-bisphosphate (FBP) concentrations, Li+ was found to be a linear noncompetitive inhibitor with respect to Mg2+. K+ was found to activate the wild-type enzyme at low concentrations (K(m) = 17 mM) and to inhibit the enzyme at high concentrations (K(IK+) = 68mM). A steady-state random ter mechanism was proposed, and a mathematical equation was derived to account for the Mg2+ and K+ kinetics and activation of FBPase. Interestingly, when Glu280 was mutated to glutamine by site-directed mutagenesis, K+ lost the ability to activate the enzyme and became a noncompetitive inhibitor with respect to Mg2+. These kinetic data suggest that K+ has two distinct sites. One is a high-affinity activation site and the other a low-affinity inhibition site. Glu280 is essential for allowing K+ to bind at the activation site. Due to the geometric constraints and its small atomic radius, Li+ can bind only at the inhibitory site. It is postulated that monovalent cations activate FBPase by helping the Arg276 residue "deshield" the partial negative charge on the 1-phosphoryl group of the substrate so that nucleophilic attack on the 1-phosphorus atom is facilitated. In addition, the monovalent cations may, along with Mg2+ ions and surrounding residues of the protein, help orient the 1-phosphoryl group so as to achieve the optimal position required for catalysis. Monovalent cations inhibit FBPase either by distorting the geometry of the active site or by retarding turnover or product release.