Perturbation of the quaternary structure and allosteric behavior of rat liver phosphofructokinase by polyethylene glycol.
The effect that polyethylene glycol (PEG) has on the aggregation of rat liver phosphofructokinase (PFK) has been directly assessed by measuring the fluorescence polarization of pyrenebutyrate covalently attached to the enzyme. After correcting for the changes in bulk viscosity that accompany PEG addition, the results confirm that PEG causes a substantial association of rat liver PFK. PEG also perturbs the linked-function kinetic parameters that describe the relationship between the allosteric inhibitor MgATP and the substrate fructose 6-phosphate (Fru-6-P). Despite the fact that PEG lowers the concentration of Fru-6-P required to produce half-maximal activity, Ka, at all concentrations of MgATP, PEG actually increases the severity of the MgATP inhibition of the enzyme by decreasing the apparent dissociation constant for MgATP in the absence of Fru-6-P and increasing the magnitude of the antagonistic coupling between MgATP and Fru-6-P. The direct effect of PEG on the inherent affinity of the enzyme for Fru-6-P in the absence of MgATP is of sufficient magnitude that the effects of the increased MgATP inhibition are overcome, resulting in net activation. By assuming that these effects arise from the aggregation induced by PEG, the relative tendency of the four enzyme forms, defined by the four limiting states of ligation, to aggregate is inferred to be Kad less than Kaxd less than or equal to Kxd much less than Kd, where Kd equals the dissociation constant for the most aggregated form of PFK dissociating to form n tetramers, and the superscripts a, x, and ax refer to the existence of bound Fru-6-P, MgATP, or both Fru-6-P and MgATP, respectively.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005636:Fructosephosphates / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011721:Pyrenes; D051381:Rats; D014783:Viscosity
D005456:Fluorescent Dyes; D005636:Fructosephosphates; D046911:Macromolecular Substances; D011721:Pyrenes; C002218:1-pyrenebutyrate; D011092:Polyethylene Glycols; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1