The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition |
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Authors: | Cabrera Ricardo Baez Mauricio Pereira Humberto M Caniuguir Andrés Garratt Richard C Babul Jorge |
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Institution: | Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. ricabrer@uchile.cl |
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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 ?. 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. |
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Keywords: | Crystal Structure Enzyme Kinetics Enzyme Mechanisms Enzyme Structure Phosphofructokinase Protein Folding Protein Structure Allosteric Regulation Analogous Enzymes Intersubunit Interactions |
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