Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2.
Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with pyrene maleimide (PM) results in a rapid inactivation of the enzyme. The loss of enzyme activity correlates with the incorporation of 2 mol of PM/mol of subunit and the concomitant dissociation of the dimeric enzyme. The two modified residues were identified as Cys-238 and Cys-295. In the presence of the negative allosteric effector, MgATP, Cys-238 was the only modified cysteine residue. Kinetic characterization of the Cys-238-labelled Pfk-2 indicates that the enzyme is fully active, with the kinetic constants ( K(m), kcat) being almost identical to the ones obtained for the native enzyme. The modified enzyme is a monomer in the absence of ligands and, like the native enzyme, behaves as a tetramer in the presence of the nucleotide. However, in the presence of fructose-6-phosphate (fru-6-P) and ATP(-4), the enzyme behaves as a dimer, suggesting that the monomers undergo re-association in the presence of the substrates and that the active species is a dimer. Modification of Pfk-2 with eosin-5-maleimide (EM) results in the labelling of Cys-295. This modified enzyme is inactive and is not able to bind to the allosteric effector, remaining as a dimer in its presence. Nonetheless, Cys-295-labelled Pfk-2 is able to bind to the substrate fru-6-P in an hyperbolic fashion with a K(d) value that is 6-fold higher than the one determined for the native enzyme. These are the first residues to be implicated in the activity and/or structure of the Pfk-2.
Baez
Mauricio
M
Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
Rodríguez
Patricio H
PH
Babul
Jorge
J
Guixé
Victoria
V
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000502:physiology*; D019281:Dimerization; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D008969:Molecular Sequence Data; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D012997:Solvents / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry
D029968:Escherichia coli Proteins; D008301:Maleimides; D012997:Solvents; D013439:Sulfhydryl Reagents; C033531:eosin maleimide; C003944:N-(3-pyrene)maleimide; D025481:Phosphofructokinase-2; D003545:Cysteine; D004801:Eosine Yellowish-(YS)