Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

Abstract

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of Kd [rabbit] and the standard free energies [.DELTA.F.degree.] of formation were calculated. The binding of Mn2+ to the enzyme (KA [association constant] = 55 .+-. 5 .times. 10-6 M; .DELTA.F.degree. = -5.75 .+-. 0.05 kcal/mol) and to the enzyme saturated with phosphoenolypyruvate (conditional free energy) (KA'' = 0.8 .+-. 0.4 .times. 10-6 M; .DELTA.F.degree. = -8.22 .+-. 0.34 kcal/mol) was measured under identical conditions giving a free energy of coupling, .DELTA.(.DELTA.F.degree.) = -2.47 .+-. 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolypyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolypyruvate binding to free enzyme (KS = 1.58 .+-. 0.10 .times. 10-4 M; .DELTA.F.degree. = -5.13 .+-. 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 .+-. 0.10 .times. 10-6 M; .DELTA.F.degree. = -8.26 .+-. 0.07 kcal/mol) also gives a large negative free energy of coupling, .DELTA.(.DELTA.F.degree.) = -3.16 .+-. 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolypyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, .DELTA.(.DELTA.F.degree.) = -0.08 .+-. 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 .+-. 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolypyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolypyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolypyruvate and with pyruvate.