This paper reports a thermodynamic method for the two-substrate nzyme-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 (ΔrHm,a anΔ Ka) but also the standard thermod ynamic properties of the ction(ΔrH_111m; ΔrG_000000000m;ΔrS_m). Microcalorimetry has been used to investigate thermod ynamics of the reversible phosphoryl transfer from ATP to creatine catalyzed by rabbit muscle-type creatine kinase (MM-CK) at different concentrations of guanidine hydrochloriΔe (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 298.15K, the standard enthalpy, Gibbs free energy, and entropy changes for the reaction at low concentrations of GuHCl were Δetermined by this metho to be 25.76kJ mol-1, 14.1kJ mol-1, and 38.9 J K-1mol-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 showeΔ that GuHCl had a noticeable inØuence on the activity of CK.