Regulation of Fructose‐Bisphosphatase Activity

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°from the R-state (compared with the 15°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.

Section: Discussionmentioning

confidence: 99%

“…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-P 2 ) to produce fructose 6-phosphate and P i (1,2). FBPase and fructose-6-phosphate-1-kinase (PFK) define a futile cycle that consumes ATP.…”

mentioning

confidence: 99%

Structures of Mammalian and Bacterial Fructose-1,6-bisphosphatase Reveal the Basis for Synergism in AMP/Fructose 2,6-Bisphosphate Inhibition

Hines

Chen

Nix

et al. 2007

“…If PEP is a physiological regulator of FBPase, then it must bind to the enzyme, but where? Observations of similar phenomena in Type III FBPases have led others to suggest competition between PEP and AMP for the same site (25), but such a mechanism is unlikely for E. coli FBPase: AMP-binding residues of porcine FBPase are present in E. coli FBPase and yet PEP has no effect on AMP inhibition of mammalian systems (2). The binding of PEP to the active site can only cause inhibition.…”

Section: Discussionmentioning

confidence: 99%

“…Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) 2 is a key enzyme of the gluconeogenic pathway, hydrolyzing fructose 1,6-bisphosphate (Fru-1,6-P 2 ) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (P i ) (1,2). In Escherichia coli, the most likely role of FBPase is the generation of fivecarbon precursors for nucleotide and polysaccharide production (3,4).…”

mentioning

confidence: 99%

Novel Allosteric Activation Site in Escherichia coli Fructose-1,6-bisphosphatase

Hines

Fromm

Honzatko

2006

Fructose-1,6-bisphosphatase (FBPase) governs a key step in gluconeogenesis, the conversion of fructose 1,6-bisphosphate into fructose 6-phosphate. In mammals, the enzyme is subject to metabolic regulation, but regulatory mechanisms of bacterial FBPases are not well understood. Presented here is the crystal structure (resolution, 1.45 Å ) of recombinant FBPase from Escherichia coli, the first structure of a prokaryotic Type I FBPase. The E. coli enzyme is a homotetramer, but in a quaternary state between the canonical Rand T-states of porcine FBPase. Phe 15 and residues at the C-terminal side of the first ␣-helix (helix H1) occupy the AMP binding pocket. Residues at the N-terminal side of helix H1 hydrogen bond with sulfate ions buried at a subunit interface, which in porcine FBPase undergoes significant conformational change in response to allosteric effectors. Phosphoenolpyruvate and sulfate activate E. coli FBPase by at least 300%. Key residues that bind sulfate anions are conserved among many heterotrophic bacteria, but are absent in FBPases of organisms that employ fructose 2,6-bisphosphate as a regulator. These observations suggest a new mechanism of regulation in the FBPase enzyme family: anionic ligands, most likely phosphoenolpyruvate, bind to allosteric activator sites, which in turn stabilize a tetramer and a polypeptide fold that obstructs AMP binding.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3. 1.3.11; FBPase) 2 is a key enzyme of the gluconeogenic pathway, hydrolyzing fructose 1,6-bisphosphate (Fru-1,6-P 2 ) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (P i ) (1, 2). In Escherichia coli, the most likely role of FBPase is the generation of fivecarbon precursors for nucleotide and polysaccharide production (3, 4). Microbial strains without FBPase (gene deletion mutants) cannot grow on gluconeogenic substrates, but thrive on glucose or other hexoses (3).FBPase and fructose-6-phosphate-1-kinase are constitutive enzymes in E. coli, and in the absence of some kind of coordinate regulation, define a futile cycle (5). Futile cycling, however, is minimal under glycolytic (6) and gluconeogenic (7) conditions, and hence FBPase must be under metabolic control. In mammals, AMP and fructose 2,6-bisphosphate (Fru-2,6-P 2 ) synergistically inhibit FBPase (8). Fru-2,6-P 2 , the concentration of which is subject to hormonal control, directly inhibits FBPase by binding to its active site, whereas AMP binds to an allosteric site (5, 9, 10) and induces a conformational change, converting the enzyme from an active quaternary conformation (R-state) to an inactive form (T-state) (11-13). In contrast, little is known regarding the regulation of FBPase in E. coli. AMP is a potent inhibitor of the E. coli enzyme (4, 14 -16), but Fru-2,6-P 2 is not present. (Fru-2,6-P 2 , however, does inhibit E. coli FBPase in assays (15, 16).) Dynamic regulation by AMP alone is unlikely, because AMP concentrations in vivo remain relatively constant under gluconeogenic and glycolytic condit...

“…FBP is important for the regulation of flux between glycolysis and gluconeogenesis (Fig. 1B); as the enzyme is inhibited by AMP and fructose 2,6-bisphosphate (Fru-2,6-P) (13). Fru-2,6-P is a competitive inhibitor with respect to the substrate Fru-1,6-P, which binds at the active site (14,15), whereas AMP binds to an allosteric site (16,17), which causes a structural transition from the active R state of the enzyme to the inactive T state (18).…”

mentioning

confidence: 99%

Inhibition of Fructose-1,6-bisphosphatase by Aminoimidazole Carboxamide Ribotide Prevents Growth of Salmonella enterica purH Mutants on Glycerol

Dougherty¹,

Boyd

Downs

2006

The enzyme fructose-1,6-bisphosphatase (FBP) is key regulatory point in gluconeogenesis. Mutants of Salmonella enterica lacking purH accumulate 5-amino-4-imidazole carboxamide ribotide (AICAR) and are unable to utilize glycerol as sole carbon and energy sources. The work described here demonstrates this lack of growth is due to inhibition of FBP by AICAR. Mutant alleles of fbp that restore growth on glycerol encode proteins resistant to inhibition by AICAR and the allosteric regulator AMP. This is the first report of biochemical characterization of substitutions causing AMP resistance in a bacterial FBP. Inhibition of FBP activity by AICAR occurs at physiologically relevant concentrations and may represent a form of regulation of gluconeogenic flux in Salmonella enterica.An efficient cellular metabolism that can adapt to changing environmental conditions requires the integration of many biochemical pathways. Subtle interactions that exist between pathways in the metabolic network are in general not well understood. One level of these interactions involves metabolites common to multiple pathways. Cellular metabolites can impact physiology through roles in transcriptional regulation, translational regulation, or by mediating allosteric effects on key enzymes. One such central metabolite with regulatory capabilities is 5-amino-4-imidazole carboxamide ribotide (AICAR).