Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus.
The binding of phosphoenolpyruvate (PEP) to the single allosteric site on phosphofructokinase (EC ) from Bacillus stearothermophilus (BsPFK) diminishes the ability of the enzyme to bind the substrate fructose 6-phosphate (Fru-6-P). Comparisons of crystal structures with either Fru-6-P or phosphoglycolate, an analog of PEP, bound have shown that Arg-162 interacts with the negatively charged Fru-6-P. Upon the binding of phosphoglycolate, Arg-162 is virtually replaced by Glu-161, which introduces a potential coulombic repulsion between enzyme and substrate [Schirmer, T. & Evans, P. R. (1990) Nature (London) 343, 140-145]. It has previously been proposed that this structural transition explains the allosteric inhibition in BsPFK, and this explanation has appeared in textbooks to illustrate how an allosteric ligand can influence substrate binding at a distance. Site-directed mutagenesis has been employed to create three mutants of BsPFK that substitute an alanine residue for Glu-161, Arg-162, or both. The E161A mutation does not affect the inhibition of BsPFK by PEP at 25 degrees C, and while the R162A mutation decreases BsPFK's affinity for Fru-6-P by approximately 30-fold, R162A diminishes the effectiveness of PEP inhibition by only 1/3. Combining E161A and R162A produces behavior comparable to R162A alone. These and other data suggest that the movement of Glu-161 and Arg-162 does not play the central role in producing the allosteric inhibition by PEP as originally envisioned in the Schirmer and Evans mechanism.
Kimmel
J L
JL
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Reinhart
G D
GD
D000494:Allosteric Regulation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D010732:Phosphofructokinase-1