31P NMR studies of the thermodynamics and kinetics of the creatine kinase reaction.

Abstract

The thermodynamic and kinetic parameters of the reaction catalyzed by creatine kinase (CK) were measured in vitro in the temperature range 13 to 35 degrees C, using 31P NMR spectroscopy, including magnetization transfer methods. The apparent equilibrium constant of the reaction and the associated enthalpy for the formation of ATP at 35 degrees C, pH 8.2, and excess [Mg2+] were 3.5 x 10(9) M-1 and -2.4 +/- 0.5 kcal/mol, respectively. The rates at equilibrium at 35 degrees C catalyzed by 1 unit/ml CK were 12.4 and 10.7 microM/s at pH 7 and 8, respectively. The rate constants per 1 unit CK/1 ml at 35 degrees C, pH 7, were 1.3 x 10(8) s-1 M-2 and 9.9 x 10(-3) s-1 M-1 in the direction of ATP and PCr formation, respectively. The activation energies in both directions were similar and corresponded to 15 +/- 2 kcal/mol at pH 7 and 17.5 +/- 1.5 kcal/mol at pH 8. Comparison of in vivo results with the above in vitro data may provide information regarding the activity and kinetics of the CK reaction.