The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

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  • Additional Information
    • Source:
      Publisher: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 0107600 Publication Model: Print Cited Medium: Print ISSN: 0014-2956 (Print) Linking ISSN: 00142956 NLM ISO Abbreviation: Eur J Biochem Subsets: MEDLINE
    • Publication Information:
      Publication: -2004: Oxford, UK : Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
      Original Publication: Berlin, New York, Springer.
    • Subject Terms:
      Adenosine Monophosphate/*pharmacology ; Enzyme Inhibitors/*pharmacology ; Fructose-Bisphosphatase/*metabolism ; Kidney/*enzymology; Allosteric Regulation ; Animals ; Binding Sites ; Enzyme Stability ; Escherichia coli ; Fructose-Bisphosphatase/antagonists & inhibitors ; Fructose-Bisphosphatase/genetics ; Fructosediphosphates/pharmacology ; Kinetics ; Magnesium/pharmacology ; Mutagenesis, Site-Directed ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Swine ; Temperature
    • Abstract:
      To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.
    • Accession Number:
      0 (Enzyme Inhibitors)
      0 (Fructosediphosphates)
      0 (Recombinant Proteins)
      415SHH325A (Adenosine Monophosphate)
      79082-92-1 (fructose 2,6-diphosphate)
      EC 3.1.3.11 (Fructose-Bisphosphatase)
      I38ZP9992A (Magnesium)
    • Publication Date:
      Date Created: 20000412 Date Completed: 20000613 Latest Revision: 20190620
    • Publication Date:
      20240829
    • Accession Number:
      10.1046/j.1432-1327.2000.01227.x
    • Accession Number:
      10759847