Isotopic probes of catalytic steps of myosin adenosine triphosphatase

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Abstract
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 32Pi 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 × 10−4 sec−1 at pH 7.4.The concentration dependencies for Pi ⇌ H18 OH exchange and for 32Pi incorporation into myosin‐bound ATP give direct measurements of the dissociation constant of Pi from S1. Both approaches show that the enzyme has a very low affinity for Pi, with an apparent Kd of > 400 mM.Measurement of the average number of water oxygens incorporated into Pi 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 Pi released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP.