Abstract
Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and AMP. AMP and Fru-2,6-P(2) bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P(2) synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P(2) in the absence of AMP. The Fru-2,6-P(2) complex exhibits a subunit pair rotation of 13 degrees from the R-state (compared with the 15 degrees rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P(2) drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P(2) synergism in mammalian FBPases. AMP and Fru-2,6-P(2) are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P(2) remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P(2) at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P(2) ligation.
Highlights
Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase; EC 3.1.3.11; FBPase)3 is a regulatory enzyme in gluconeogenesis, cleaving the 1-phosphoryl group from fructose 1,6-bisphosphate (Fru-1,6-P2) to produce fruc
Fru-2,6-P2 binds to the active site and inhibits catalysis competitively with respect to Fru-1,6-P2 (6 –9), whereas AMP binds to allosteric sites separated by no less than 28 Å from the nearest active site [10]
The conformational change in the dynamic loop is consistent with competitive inhibition of catalysis by AMP with respect to Mg2ϩ [28]
Summary
Materials—Fru-1,6-P2, Fru-2,6-P2, NADPϩ, DEAE-Sepharose, and Cibacron Blue-Sepharose came from Sigma. Crystals of E. coli FBPase with citrate and Fru-2,6-P2 grew from droplets containing 2 l of a protein solution (15 mg/ml enzyme, 20 mM dithiothreitol, 0.1 mM EDTA, 5 mM Fru1,6-P2, 5 mM Fru-2,6-P2, 5 mM MgCl2) and 2 l of a precipitant solution (50 mM sodium citrate, pH 5.3, 25% (w/v) polyethylene glycol 1500, and 20% (w/v) sucrose). 2,6-P2 complex of porcine FBPase grew from droplets containing 2 l of a protein solution (10 mg/ml enzyme, 0.2 mM EDTA, 5 mM Fru-2,6-P2, 5 mM MgCl2) and 2 l of a precipitant solution (8.5% (w/v) polyethylene glycol 3350, 5% (v/v) t-butanol, 27%. Crystals of the Zn2ϩ1⁄7Fru-2,6-P2 complex of porcine FBPase grew from droplets containing 2 l of a protein solution (10 mg/ml enzyme, 5 mM Fru-2,6-P2, 2 mM ZnCl2) and 2 l of a precipitant solution (10% (w/v) polyethylene glycol 3350, 5% (v/v) t-butanol, 27%. Structural models underwent energy minimization followed by individual thermal parameter refinement using CNS [46]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
