RGD Reference Report - Dominant negative role of the glutamic acid residue conserved in the pyruvate kinase M(1) isozyme in the heterotropic allosteric effect involving fructose-1,6-bisphosphate. - Rat Genome Database

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Dominant negative role of the glutamic acid residue conserved in the pyruvate kinase M(1) isozyme in the heterotropic allosteric effect involving fructose-1,6-bisphosphate.

Authors: Ikeda, Y  Taniguchi, N  Noguchi, T 
Citation: Ikeda Y, etal., J Biol Chem. 2000 Mar 31;275(13):9150-6. doi: 10.1074/jbc.275.13.9150.
RGD ID: 151708737
Pubmed: PMID:10734049   (View Abstract at PubMed)
DOI: DOI:10.1074/jbc.275.13.9150   (Journal Full-text)

Pyruvate kinase M(1), a nonallosteric isozyme, lacks heterotropic allosteric effect involving fructose-1,6-bisphosphate (FBP). To explore the molecular basis for this, a series of mutants were prepared and characterized, in which the possible candidate, Glu-432, was replaced in the rat M(1) isozyme and its allosteric mutant with the replacement of Ala-398 by Arg. Although these single mutants of Glu-432 remained nearly fully active, similar to the wild type, only the mutants with replacements by Lys and Ala were more efficiently activated by FBP when the enzymes were inhibited by L-phenylalanine. Kinetic analyses and ligand-induced fluorescence quenching studies using the allosteric double mutants indicated that the loss of a negative charge at residue 432 led to a dramatic decrease in the apparent activation constant and apparent K(d) for FBP. Furthermore, this enhancement was found to be associated with the modification of the FBP-binding site rather than the alteration of the subunit assembly. These findings suggest that Glu-432 hinders the heterotropic allosteric effect by preventing the binding of FBP through a repulsive electrostatic interaction and thereby contributes to its unique unregulated properties, independent of the shifted allosteric transition.



Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
PkmRatprotein homotetramerization  IDA  RGD 
PkmRatpyruvate biosynthetic process  IDA  RGD 

Molecular Function

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
PkmRatpyruvate kinase activity  IDA  RGD 

Objects Annotated

Genes (Rattus norvegicus)
Pkm  (pyruvate kinase M1/2)


Additional Information