The Role of Cations in Yeast Phosphofructokinase Catalysis

Abstract

Abstract The catalytic activity of yeast phosphofructokinase is dependent on the concentrations of both uncomplexed Mg2+ and MgITP2-, and not on the concentration of uncomplexed ITP. The allosteric inhibition of catalysis by ATP is dependent on the concentration of MgATP2- and not on the concentration of uncomplexed ATP. It is proposed that the nucleoside triphosphate substrates are bound as Mg2+ complexes, that catalysis requires uncomplexed Mg2+, and that MgATP2- is the negative effector. Addition of NH4Cl 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 NH4+-enzyme complexes. Formation of the stronger complex, which has an apparent dissociation constant of 3 mm, 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, MgATP2-. The apparent dissociation constant of the weaker NH4+-enzyme complex increases as the concentration of the positive effector decreases or the concentration of negative effector increases, or both. The presence of 50 mm NH4Cl 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 NH4+ 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 mm, 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 mm 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 NH4+ and K+ sites are distinct from each other and that the catalytic response to the binding of NH4+ and K+ to these sites is not additive.