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The ATP in equilibrium with Pi exchange of actin-activated myosin subfragment 1 was investigated as a function of actin concentration. The maximum rate of exchange was 1.7 x 10(-3) s-1 at a Pi concentration of 2 mM, and this rate was independent of medium ADP concentration. The observed rate is 50 times faster than expected if reversal starts from the AM.ADP state that can be formed from medium ADP. The existence of a preceding higher energy AM'.ADP state which exists in significant concentration only during the steady-state hydrolysis of ATP accounts for this discrepancy and also for the independence of the rate of in equilibrium with Pi exchange on medium ADP concentration. Addition of ADP and Pi to myosin results in the formation of an equilibrium amount of enzyme-bound ATP. The inhibitory effect of actin on the formation of enzyme-bound ATP has been investigated and a value of 4 x 10(6) M-1 has been determined for the binding constant of actin to M*.ADP and an upper limit of 2 x 10(4) M-1 has been determined for the binding constant of actin to M*.ATP.
Sleep et al. (Tue,) studied this question.