Unfolding pathway of the dimeric and tetrameric forms of phosphofructokinase-2 from Escherichia coli.
Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds of interfaces: a monomer-monomer interface, critical for enzymatic activity, and a dimer-dimer interface formed upon tetramerization due to allosteric binding of MgATP. In this work, Pfk-2 was denatured by guanidine hydrochloride (GdnHCl) and the impact of ligand binding on the unfolding pathway of the dimeric and the tertrameric forms of the enzyme was examined. The unligated dimeric form unfolds and dissociates from 0.15 to 0.8 M GdnHCl without the accumulation of native monomers, as indicated by circular dichroism and size exclusion chromatography measurements. However, a monomeric intermediate with an expanded volume and residual secondary structure accumulates above 0.8 M GdnHCl. The dimeric fructose-6-P-enzyme complex shows a shift in the simultaneous dissociation and unfolding process to elevated GdnHCl concentrations (from 0.8 to 1.4 M) together with the expulsion of the ligand detected by intrinsic fluorescence measurements. The unfolding pathway of the tetrameric MgATP-enzyme complex shows the accumulation of a tetrameric intermediate with altered fluorescence properties at about 0.4 M GdnHCl. Above this concentration a sharp transition from tetramers to monomers, without the accumulation of either compact dimers or monomers, was detected by light scattering measurements. Indeed, the most populated species was a partially unfolded monomer about 0.7 M GdnHCl. On the basis of these results, we suggest that the subunit contacts are critical for the maintenance of the overall structure of Pfk-2 and for the binding of ligands, explaining the reported importance of the dimeric state for enzymatic activity.
Baez
Mauricio
M
Departamento de Biología, Facultad de Ciencias Universidad de Chile, Casilla 653, Santiago, Chile.
Cabrera
Ricardo
R
Guixé
Victoria
V
Babul
Jorge
J
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism; D008027:Light; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; D005636:Fructosephosphates; C027132:1-anilino-8-naphthalenesulfonate; C027618:fructose-6-phosphate; D025481:Phosphofructokinase-2; D017301:Ca(2+) Mg(2+)-ATPase