Dynamic interaction between actin and myosin subfragment 1 in the presence of ADP

The equilibrium and dynamics of the interaction between actin, myosin subfragment 1 (S1), and ADP have been investigated by using actin which has been covalently labeled at Cys-374 with a pyrene group. The results are consistent with actin binding to S1.ADP (M.D) in a two-step reaction, A + M.D K1 equilibrium A-M.D K2 equilibrium A.M.D, in which the pyrene fluorescence only monitors the second step. In this model, K1 = 2.3 X 10(4) M-1 (k+1 = 4.6 X 10(4) M-1 s-1) and K2 = 10 (k+2 less than or equal to 4 s-1); i.e., both steps are relatively slow compared to the maximum turnover of the ATPase reaction. ADP dissociates from both M.D and A-M.D at 2 s-1 and from A.M.D at greater than or equal to 500 s-1; therefore, actin only accelerates the release of product from the A.M.D state. This model is consistent with the actomyosin ATPase model proposed by Geeves et al. (1984) J. Muscle Res. Cell Motil. 5, 351. The results suggest that A-M.D cannot break down at a rate greater than 4 s-1 by dissociation of ADP, by dissociation of actin, or by isomerizing to A.M.D. It is therefore unlikely to be significantly occupied in a rapidly contracting muscle, but it may have a role in a muscle contracting against a load where the ATPase rate is markedly inhibited. Under these conditions, this complex may have a role in maintaining tension with a low ATP turnover rate.