Isotopic probes of catalytic steps of myosin adenosine triphosphatase

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 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 × 10⁻⁴ sec⁻¹ at pH 7.4. The concentration dependencies for P_i ⇌ H¹⁸ OH exchange and for ³²P_i 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 > 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.