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.
Highlights
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
In Escherichia coli, the glycolytic step of the ATP-dependent phosphorylation of fructose-6-P is catalyzed by two different phosphofructokinases, Pfk-1 and Pfk-2
The kinetic mechanism of Pfk-2 has been characterized to consist of an ordered sequential addition of substrates where fructose-6-P binding to the active site is required for the catalytic binding of MgATP
Summary
Crystallization and Data Collection—The enzyme was purified according to the method described by Parducci et al [15]. Crystals were grown by the vapor diffusion method in a hanging drop consisting of 2 l of the protein stock solution (2 mg/ml of protein in 25 mM Tris-HCl pH 7.6 buffer, 10 mM MgCl2, 30 mM DTT, and 6 mM fructose-6-P) and 2 l of a reservoir solution comprising 23% PEG 4000, 100 mM sodium acetate, pH 4.75, and 200 mM ammonium acetate. X-ray diffraction data were collected at 100 K. Structure Solution and Refinement—The structure of E. coli Pfk-2 in complex with fructose-6-phosphate was solved by TABLE 1 Data collection parameters and structure refinement statistics The values in parentheses are for the outer resolution shells
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