title: Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.
abstract: We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, England.
authors.forename: S A
authors.initials: SA
authors.lastname: Berger
authors.forename: P R
authors.initials: PR
authors.lastname: Evans
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000502:physiology*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005636:Fructosephosphates / Q000737:chemistry; D007700:Kinetics; D010728:Phosphoenolpyruvate / Q000737:chemistry; D010732:Phosphofructokinase-1 / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D005636:Fructosephosphates; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1