Crystal Complex of Phosphofructokinase-2 of Escherichia coli with Fructose-6-phosphate: KINETIC AND STRUCTURAL ANALYSIS OF THE ALLOSTERIC ATP INHIBITION

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 delay...

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Bibliographic Details
Published in:The Journal of biological chemistry 2011-02, Vol.286 (7), p.5774-5783
Main Authors: Cabrera, Ricardo, Baez, Mauricio, Pereira, Humberto M, Caniuguir, Andrés, Garratt, Richard C, Babul, Jorge
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - chemistry
Adenosine Triphosphate - metabolism
Allosteric Regulation
Catalytic Domain
Crystallography, X-Ray
Escherichia coli
Escherichia coli - enzymology
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - metabolism
Evolution, Molecular
Fructosephosphates - chemistry
Fructosephosphates - metabolism
Kinetics
Phosphofructokinase-1 - chemistry
Phosphofructokinase-1 - metabolism
Phosphofructokinase-2 - chemistry
Phosphofructokinase-2 - metabolism
Protein Structure and Folding
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Summary: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 K₀.₅ for fructose-6-P and a decrease in the apparent kcat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (nH 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M110.163162