Energetics and kinetics of the interconversion of two myosin subfragment-1.cntdot.adenosine 5'-diphosphate complexes as viewed by phosphorus-31 nuclear magnetic resonance

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Abstract
The 31P NMR spectrum of MgADP bound to myosin subfragment-1 (S-1) from rabbit skeletal muscle at 0.degree. C contains 2 resolved .beta.-phosphate resonances corresponding to 2 interconvertible conformations of the S-1.cntdot.ADP complex. The 2 conformations, MT*ADP and MR*ADP, are in slow exchange on the NMR time scale, and the rates of interconversion are < 20 s-1. This is consistent with transient kinetic experiments reported in the literature and allows a determination of the rate constants of interconversion: k+ .apprxeq. k- .apprxeq. 7 s-1 at 0.degree. C. The relative population of the 2 conformations is highly temperature dependent and only 1 form is significantly populated at 25.degree. C. Simulations of the 31P NMR spectra are used to evaluate an equilibrium constant at various temperatures from 0.degree.-25.degree. C. The standard enthalpy and entropy differences for the R .fwdarw. T transition are determined from the variation of the relative free energies of the 2 states as a function of temperature: .DELTA.H.degree. [enthalpy] = 15 (.+-. 2) kcal/mol and .DELTA.S.degree. = 55 (.+-. 5) cal/(degree mol) (K = 1 at 271.degree. K). This suggests that a significant conformational change occurs in the R-T transition with MgADP bound in the active site. The entropy and enthalpy differences are nearly compensatory at physiological temperatures. At 25.degree. C the endothermic R .fwdarw. T transition is entropy driven, and .DELTA.G.degree. [Gibbs free energy] = 1.4 kcal/mol.