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.
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.
Ikeda
Y
Y
Department of Biochemistry, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan.
Taniguchi
N
N
Noguchi
T
T
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005635:Fructosediphosphates / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005635:Fructosediphosphates; D007527:Isoenzymes; D011994:Recombinant Proteins; D018698:Glutamic Acid; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate