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J Biol Chem. 2011 Feb 18;286(7):5774-83. doi: 10.1074/jbc.M110.163162. Epub 2010 Dec 8.

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition.

Author information

  • 1Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. ricabrer@uchile.cl

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.

PMID:
21147773
[PubMed - indexed for MEDLINE]
PMCID:
PMC3037690
Free PMC Article

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