Investigation of allosteric regulation of porcine fructose-1,6-bisphosphatase
Porcine Fructose-1,6-bisphosphatase is a homotetramer with four identical subunits. It plays a central role in gluconeogenesis and is tightly regulated by metabolites fructose 2,6-bisphosphate (F26P2) and AMP. Loop 52-72, loop 182-194 and residues 7-11 of the N-terminal segment play important role in the mechanism of catalysis and allosteric inhibition by AMP by retaining a structural hydrophobic region. Disruption of the hydrophobic region will evidently affect the catalysis and regulation of FBPase. Mutation of Thr53 reduced the catalysis activity of FBPase, mutation of Thr194 eliminated inhibition of AMP due to its low affinity to the enzyme, while double mutation Thr53/194 had accumulative effect. Mutation Ala10 exhibited biphasic AMP inhibition, an AMP high affinity site which had comparable IC50 with wild-type FBPase and an AMP low affinity site which required 3000-fold AMP to reach 50% relative inhibition.