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Effect of Buffer Solutions on Activation of Shamouti Orange Pyrophosphate‐Dependent Phosphofructokinase by Fructose 2,6‐Bisphosphate
Author(s) -
Van Praag Esther,
Tzur Assaf,
Zehavi Uri,
Goren Raphael
Publication year - 2000
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216540050022485
Subject(s) - chemistry , phosphofructokinase , chloride , tris , hepes , fructose 2,6 bisphosphate , pyrophosphate , substrate (aquarium) , medicinal chemistry , fructose , enzyme , inorganic chemistry , nuclear chemistry , glycolysis , biochemistry , organic chemistry , oceanography , geology
Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6‐bisphosphate (Fru‐2,6‐P2) in the glycolytic reaction. The effect of activation by Fru‐2,6‐P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 plus/minus 0.3 nM in Hepes‐NaOH and 7.75 plus/minus 1.49 nM in Tris‐HCl. The presence of chloride ions (39 mM) in the Tris‐HCl buffer inhibits PFP. Indeed, when using a Hepes‐NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 plus/minus 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru‐2,6‐P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru‐1,6‐P2 from 200 plus/minus 15.6 muM in absence of Fru‐2,6‐P2 to 89 plus/minus 10.3 muM in its presence (10 muM). In the presence of chloride (39 mM), the affinity for the substrate decreased with Km = 150 plus/minus 14 muM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.