A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [32P] - ATP formed from 32P(i) by S1 catalysis and the amount of rapidly formed [32P] - ATP tightly bound to S1, the value of the off constant is approximately 2.8 X 10(-4) sec -1 at pH 7.4. The concentration dependencies for P(i) in equilibrium H18 OH exchange and for (32)P(j) incorporation into myosin-bound ATP give direct measurements of the dissociation constant of P(i) from S1. Both approaches show that the enzyme has a very low affinity for P(i), with an apparent K(d) of greater than 400 mM. Measurement of the average number of water oxygens incorporated into P(i) released from ATP by S1-catalyzed hydrolysis in the presence of Mg2+ suggests that the hydrolytic step reverses an average of at least 5.5 times for each ATP cleaved. With the Ca2+ -activated hydrolysis, less than one oxygen from water appears in each P(i) released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP.
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