Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties.
Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer-tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern-Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP-enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity.
Guixé
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Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
D020106:Acrylamide / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D008301:Maleimides / Q000378:metabolism / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000378:metabolism* / Q000494:pharmacology
D005456:Fluorescent Dyes; D005636:Fructosephosphates; D008024:Ligands; D008301:Maleimides; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D020106:Acrylamide; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; C003944:N-(3-pyrene)maleimide; D010732:Phosphofructokinase-1