KINETIC AND EQUILIBRIUM-ANALYSIS OF THE INTERACTIONS OF ACTOMYOSIN SUBFRAGMENT-1.ADP WITH BERYLLIUM FLUORIDE

The hypothesis that the stable ternary complex formed between myosin s ubfragment-1, MgADP and beryllium fluoride (BeF3-), denoted S-1not-equ al.ADP.BeF3-, is an analog of the intermediate state S-1*.ADP.P(i) ha s been tested in this work by examining the interactions of S-1not-equ al.ADP.BeF3- with actin. Equilibrium binding measurements revealed tha t actin bound weakly to the S-1not-equal.ADP.BeF3- complex (K(a) = 10( 4) M-1) in the presence of 40 mM KCl. The stability of this complex wa s strongly salt-dependent. The association constant of BeF3- to the ac to-S-1.ADP complex (K(Be) approximately 10(3) M-1) was 100-fold weaker than its binding to the S-1.ADP complex. While inhibiting the S-1 ATP ase strongly, BeF3- had no effect on the V(max) value (10 +/- 1.0 s-1) of the actin-activated ATPase of S-1. The rates of BeF3- binding and dissociation from the acto-S-1.ADP.BeF3- complex were determined by st opped-flow measurements. The hyperbolic dependence of the rates of BeF 3- binding to acto-S-1-ADP (k(obs)) on BeF3- concentrations suggested that the acto-S-1.ADP.BeF3- complex was formed in at least two steps: binding followed by isomerization. The binding Constant was 1.2 x 10(3 ) M-1, and the maximum k(obs) was 2.5 s-1. The dissociation of BeF3- f rom the acto-S-1.ADP.BeF3- complex was monitored via decrease in the f luorescence of 1-N6-ethenoadenosine diphosphate (epsilonADP). The fluo rescence decrease fitted two exponential terms. A kinetic scheme which is consistent with earlier results on the interactions of S-1.ADP wit h BeF3- (Phan & Reisler, 1992) and accounts for two exponential terms involves an equilibrium between two isomerized states S-1not-equal.ADP .BeF3-and S-1not-equal not-equal.ADP.BeF3-. The rate constant for the dissociation of BeF3- from the S-1not-equal.ADP.BeF3- complex was incr eased between 10(4)- and 10(5)-fold by actin. These results show that the AS-1not-equal.ADP.BeF3- complex has properties similar to those of the intermediate state AS-1*.ADP.P(i) and thus support the hypothesi s that S-1not-equal.ADP.BeF3- is a good analog of the S-1*-ADP-P(i) s tate.