Role of Cys‐295 on subunit interactions and allosteric regulation of phosphofructokinase‐2 from Escherichia coli

Abstract

In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k cat value and an increment in the K m values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.

Crystallographic Structure of Phosphofructokinase-2 from Escherichia coli in Complex with Two ATP Molecules. Implications for Substrate Inhibition

“…27 A homodimer is observed in the asymmetric unit, which makes extensive contacts with a symmetry-related mate (Fig. 2), forming what appears to be the tetramer under the abovementioned conditions.…”

Mutantelec: An In Silico mutation simulation platform for comparative electrostatic potential profiling of proteins

“…Site‐directed mutagenesis is a standard experimental technique used to generate site‐specific mutations in known protein‐coding genes . By deleting or substituting particular residues, the role of individual amino acids in the reaction mechanisms of enzymes or in the structural configuration of proteins can be studied . However, this technique is very laborious and time consuming, and in silico tools to help direct the selection of candidate residues are scarce.…”

“…[18] By deleting or substituting particular residues, the role of individual amino acids in the reaction mechanisms of enzymes or in the structural configuration of proteins can be studied. [19][20][21][22][23] However, this technique is very laborious and time consuming, and in silico tools to help direct the selection of candidate residues are scarce.…”

Reversible unfolding of dimeric phosphofructokinase‐2 fromEscherichia colireveals a dominant role of inter‐subunit contacts for stability

“…Light scattering and gel filtration analyses [12,13] have shown that E. coli Pfk‐2 is primarily a dimer in solution, which quaternary structure is required for enzymatic activity [12,14]. As shown by its crystal structure (PDB ID code 3CQD), each subunit can be divided in two parts: the conserved α/β/α domain and the additional β‐sheet structural element that protrudes from it.…”