Microcalorimetric studies on the creatine kinase-catalyzed reaction in the presence of guanidine hydrochloride

Abstract

This paper reports a thermodynamic method for the two-substrate enzyme-catalyzed reaction by a random sequential mechanism in the presence of a chemical denaturant. This is a convenient method to produce not only the apparent molar thermodynamic constants (Δ r H m,a and K a) but also the standard thermodynamic properties of the reaction (Δ r H m ⊕,Δ r G m ⊕,Δ r S m ⊕). Microcalorimetry has been used to investigate thermodynamics of the reversible phosphoryl transfer from ATP to creatine catalyzed by rabbit muscle-type creatine kinase (MM-CK) at different concentrations of guanidine hydrochloride (GuHCl). From a thermodynamic viewpoint, this enzyme-catalyzed reaction follows a rapid-equilibrium, random mechanism, i.e. the chemical steps are slower than those for binding of reagents, and there is no obligatory order of binding or release. At 298.15 K, the standard enthalpy, Gibbs free energy, and entropy changes for the reaction at low concentrations of GuHCl were determined by this method to be 25.76 kJ mol −1, 14.1 kJ mol −1, and 38.9 J K −1 mol −1, respectively, in agreement with those in the absence of GuHCl. The experimental results demonstrated the reliability of the above thermodynamic method, and indicated that inactivation of CK by low concentrations of GuHCl had no effect on the standard thermodynamic parameters for the CK-catalyzed reaction. A novel method for the determination of creatine kinase activity, the microcalorimetric assay for CK, was also proposed in this paper. The experimental results showed that GuHCl had a noticeable influence on the activity of CK.