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

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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°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°C, pH 8.2, and excess [Mg2+] were 3.5 × 109 M−1 and −2.4 ± 0.5 kcal/mol, respectively. The rates at equilibrium at 35°C catalyzed by 1 unit/ml CK were 12.4 and 10.7 μM /s at pH 7 and 8, respectively. The rate constants per 1 unit CK/ 1 ml at 35°C, pH 7, were 1.3 × 108 s−1M−2 and 9.9 × 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.