title: The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition.
abstract: Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K(0.5) for fructose-6-P and a decrease in the apparent k(cat) as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n(H) of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. ricabrer@uchile.cl
authors.forename: Ricardo
authors.initials: R
authors.lastname: Cabrera
authors.forename: Mauricio
authors.initials: M
authors.lastname: Baez
authors.forename: Humberto M
authors.initials: HM
authors.lastname: Pereira
authors.forename: Andrés
authors.initials: A
authors.lastname: Caniuguir
authors.forename: Richard C
authors.initials: RC
authors.lastname: Garratt
authors.forename: Jorge
authors.initials: J
authors.lastname: Babul
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000494:Allosteric Regulation; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D019143:Evolution, Molecular; D005636:Fructosephosphates / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000378:metabolism; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005636:Fructosephosphates; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D025481:Phosphofructokinase-2; D010732:Phosphofructokinase-1