Enthalpy changes for intermediate steps of the ATP hydrolysis catalyzed by myosin subfragment-1.

The heat production during the ATP hydrolysis catalyzed by myosin subfragment 1 in 0.1 M KC1 containing 0.01 M MgClz and 0.02 M Tris/HCl (pH 7.8) at 23°C was followed using a microcalorimeter with an improved time resolution. The results indicate that: (a) the binding of ATP to Subfragment 1 is exothermic (AH = -90 kJ mol-‘, all the AH values are corrected for protonation heat of Tris). (b) The actual cleavage of ATP on Subfragment 1 is endothermic (AH = +83 kJ mol-I), which contrasts to the situation in free solution where ATP hydrolysis is exothermic, AH = -23 kJ mol-‘. Thus binding and cleavage together, which form the myosin+product complex at a rate which greatly exceeds the rate of decay of the complex (Pi release), is marginally exothermic (AH = -7 kJ mol-‘). (c) The slow Pi release is strongly exothermic (AH = -88 kJ mol-‘), as previously suggested by Yamada et al. (Yamada, T., Shimizu, H., and Suga, H. (1973) Biochim. Biophys. Acta 305, 643-653). (d) The dissociation of ADP from Subfragment 1 is endothermic (AH = +72 kJ mol-‘), being consistent with our recent result on the ADP binding to Subfragment 1 and heavy meromyosin (Kodama, T., Watson, I. D., and Woledge, R. C. (1977) J. Biol. Chem. 252,8085-8087). Thus in the myosin-catalyzed hydrolysis of ATP large enthalpy changes, as compared with the enthalpy change for the overall reaction, accompany several intermediate steps of the enzyme catalytic cycle. This is probably connected with the energy transduction role of myosin in muscle contraction.