Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli.
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.
Caniuguir
Andrés
A
Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Cabrera
Ricardo
R
Báez
Mauricio
M
Vásquez
Claudio C
CC
Babul
Jorge
J
Guixé
Victoria
V
D000494:Allosteric Regulation; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D025481:Phosphofructokinase-2 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005636:Fructosephosphates; D021122:Protein Subunits; C027618:fructose-6-phosphate; D025481:Phosphofructokinase-2; D019791:Guanidine; D003545:Cysteine