Kinetic and Thermodynamic Properties of the Ternary Complex between F‐actin, Myosin Subfragment 1 and Adenosine 5′‐[β,γ‐imido]triphosphate

Abstract

Equilibrium constants for the formation of a ternary complex between [rabbit muscle] actin, myosin subfragment 1 (S1) and the nonhydrolyzable ATP analog adenosine 5''-[.beta.,.gamma.-imido]triphosphate (AdoPP[NH]P) were determined from light-scattering titrations under a variety of conditions. The affinities of S1 (binding constant K1) and acto .cntdot. S1 (K4) for AdoPP[NH]P have relatively low dependencies on temperature (.DELTA.H.degree. .apprxeq. -15 to 30 kJ mol-1) and ionic strength, in contrast to the affinities of S1 (K2) and S1 .cntdot. AdoPP[NH]P (K3) for actin which are influenced quite strongly by temperature (.DELTA.H.degree. .apprxeq. 50-65 kJ mol-1) and ionic strength; K2 decreases by a factor of 10-15 between I [ionic strength] = 0.05 M and I = 0.2 M and K3 decreases by a factor of 5. K1 and, by detailed balance, K2 as well were .apprx. 10 times higher than hitherto reported values (K1 = 3.4 .times. 107 M-1, K2 = 6 .times. 10-8 M-1, at 24.degree. C, I = 0.09 M, pH 8.0). The binding of ADP to S1 is .apprx. 10-fold weaker than that of AdoPP[NH]P, being however much more exothermic (.DELTA.H.degree. = 70 kJ mol-1 at I = 0.1 M) and having a negative standard entropy change (.DELTA.S.degree. = -125 J mol-1 K-1), in contrast to AdoPP[NH]P binding for which the calculated .DELTA.S.degree. has positive values. The observed rate constant of dissociation of acto .cntdot. S1 by AdoPP[NH]P showed an almost hyperbolic dependence on the nucleotide concentration, reaching a maximum of 15 s-1 at I = 0.055 M and 5 s-1 at I = 0.275 M pH 8.0, 23.degree. C; at 5.degree. C this value was somewhat higher. The rate constant of dissociation of AdoPP[NH]P from its complex with acto .cntdot. S1 was estimated to exceed 400 s-1 at 23.degree. C and to be of the order of 150 s-1 at 4.degree. C. The observed rate constant for the association of the S1 .cntdot. nucleotide complex and actin was proportional to actin concentrations up to 60 .mu.M, thus defining an apparent 2nd-order rate constant of 2 .times. 104 M-1 s-1 at I = 0.125 M and 23.degree. C. A reaction scheme is proposed in which isomerizations of the acto .cntdot. S1 and acto .cntdot. S1 .cntdot. nucleotide complexes can occur.