title: The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus.
abstract: 6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V(max), depending on the lipid concentration and vesicle composition, but no change in K(m). Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.
authors.affiliation: School of Chemistry, University of Southampton, Southampton, UK.
authors.forename: Maria-Nefeli
authors.initials: MN
authors.lastname: Tsaloglou
authors.forename: George S
authors.initials: GS
authors.lastname: Attard
authors.forename: Marcus K
authors.initials: MK
authors.lastname: Dymond
mesh_terms: D055162:Biocatalysis; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D019301:Oleic Acid; D010713:Phosphatidylcholines; D010732:Phosphofructokinase-1 / Q000378:metabolism*
chemical_list: D008055:Lipids; D008081:Liposomes; D010713:Phosphatidylcholines; D019301:Oleic Acid; D010732:Phosphofructokinase-1; C017251:1,2-oleoylphosphatidylcholine