Interactions between citrate and nucleoside triphosphates in binding to phosphofructokinase.
Interrelationships between the binding by rabbit muscle phosphofructokinase of citrate, ATP, GTP, and adenyl-5'-yl imidodiphosphate (AMP-PNP) were investigated. To allow measurements at 25 degrees C, pyruvate kinase and phosphoenolpyruvate were included in the dialysis media to rephosphorylate ADP formed by the weak ATPase action of phosphofructokinase. Binding of citrate was enhanced by GTP nearly as much as by ATP, although GTP does not inhibit the catalytic activity of the enzyme. The results are consistent with the interpretation that binding of GTP, and, by analogy, ATP, at the catalytic site enhances the binding of citrate. AMP-PNP also enhanced citrate binding. Both ATP and GTP appear to bind at three sites per enzyme subunit, with the apparent third site binding relatively weakly. The estimated dissociation constants for the first two sites, about 33 microM for both for ATP compared with 3 and 280 microM for GTP, are consistent with kinetic results that imply lack of effective competition by GTP for the inhibitory site. When a compound binds at two or more sites on a macromolecule, the position and shape of the binding curve are sensitive to the geometric mean of the binding constants but quite insensitive to the magnitudes of the individual constants; thus, binding affinities cannot be estimated with confidence in such cases.
Ogawa
Y
Y
Atkinson
D E
DE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D002951:Citrates / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D002250:Carbon Radioisotopes; D002951:Citrates; D000266:Adenylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1