PMID:12927023
| Citation |
Baez, M, Rodríguez, PH, Babul, J and Guixé, V (2003) Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2. Biochem. J. 376:277-83 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC1223755 Online version:10.1042/BJ20030795 |
| Keywords |
Allosteric Regulation; Amino Acid Sequence; Cysteine/chemistry; Cysteine/physiology; Dimerization; Eosine Yellowish-(YS)/analogs & derivatives; Eosine Yellowish-(YS)/chemistry; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Kinetics; Maleimides/chemistry; Molecular Sequence Data; Phosphofructokinase-2/chemistry; Phosphofructokinase-2/metabolism; Protein Structure, Quaternary; Solvents/chemistry; Sulfhydryl Reagents/chemistry |
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