The Role of Cations in Yeast Phosphofructokinase Catalysis

The catalytic activity of yeast phosphofructokinase is dependent on the concentrations of both uncomplexed Mg 2+ and MgITP 2- , and not on the concentration of uncomplexed ITP. The allosteric inhibition of catalysis by ATP is dependent on the concentration of MgATP 2- and not on the concentration of uncomplexed ATP. It is proposed that the nucleoside triphosphate substrates are bound as Mg 2+ complexes, that catalysis requires uncomplexed Mg 2+ , and that MgATP 2- is the negative effector. Addition of NH 4 Cl increases the catalytic activity of yeast phosphofructokinase up to 20-fold depending on the allosteric state of the enzyme. This activation can be resolved into the formation of two NH 4 + -enzyme complexes. Formation of the stronger complex, which has an apparent dissociation constant of 3 m m , produces about a 100% increase in the rate of catalysis irrespective of the allosteric state of the enzyme. Formation of the weaker complex is only detected catalytically in the presence of low concentrations of the positive effector, fructose-6-P, or in the presence of the negative effector, MgATP 2- . The apparent dissociation constant of the weaker NH 4 + -enzyme complex increases as the concentration of the positive effector decreases or the concentration of negative effector increases, or both. The presence of 50 m m NH 4 Cl increases the affinity of the enzyme for the substrate fructose-6-P and decreases the Hill coefficient of fructose-6-P. It was concluded that NH 4 + functions as an activator when binding to the stronger site and as a positive allosteric effector when binding to the weaker site. Addition of KCl also increases the catalytic activity of the enzyme. This activation can be resolved into the formation of two K + -enzyme complexes. Formation of the stronger complex, which has an apparent dissociation constant of about 10 m m , produces a 50% increase in catalytic activity and does not appear to alter the allosteric state of the enzyme. Although the apparent dissociation constant of the weaker K + -enzyme complex increases as the initial concentration of positive effector decreases or negative effector increases (or both), the presence of 200 m m KCl does not change the affinity of the enzyme for fructose-6-P or the Hill coefficient of this substrate. It was concluded therefore that K + is not a significant allosteric effector. Competition experiments indicate that the stronger NH 4 + and K + sites are distinct from each other and that the