Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate.
The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress.
Podestá
Florencio E
FE
Centro de Estudios Fotosintéticos y Bioquímicos, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Argentina. fpodesta@fbioyf.unr.edu.ar
Plaxton
William C
WC
D001667:Binding, Competitive; D002384:Catalysis; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000737:chemistry / Q000378:metabolism*; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D035281:Plant Tubers / Q000737:chemistry / Q000201:enzymology*; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D005635:Fructosediphosphates; D008024:Ligands; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; C029063:fructose-1,6-diphosphate