Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2

Biochem J. 2003 Nov 15;376(Pt 1):277-83. doi: 10.1042/BJ20030795.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Escherichia coli Proteins
  • Maleimides
  • Solvents
  • Sulfhydryl Reagents
  • eosin maleimide
  • N-(3-pyrene)maleimide
  • Phosphofructokinase-2
  • Cysteine
  • Eosine Yellowish-(YS)