Subunit-subunit interactions in TF₁ as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M^-1s^-1. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M^-1 · s^-1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A _-1 = 3 · 10^-3 s^-1) and a slower second phase (k^A _-2 < 0.2 · 10^-3 s^-1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.