EFFECT OF TEMPERATURE ON THE CREATINE-KINASE EQUILIBRIUM

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as K' = [SIGMA-ATP][Cr]/[SIGMA-ADP][SIGMA-PCr] The symbol SIGMA-denotes the sum of all the ionic and metal complex species of the reactant components in m. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5-degrees-C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (DELTA-H'degrees) was calculated from a van't Hoff plot of log10.K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (DELTA-S'degrees) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both DELTA-H'degrees and DELTA-S'degrees are independent of temperature for the creatine kinase reaction, and that DELTA-C(p)'degrees, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38-degrees-C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (DELTA-G'degrees) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T-DELTA-S'degrees term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. PCr2- + ADP3- + H+ = ATP4- + Cr (2) MgPCr + MgADP1- + H+ = MgATP2- + Cr + Mg2+ (3) The DELTA-H-degrees for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38-degrees-C. The corresponding DELTA-S-degrees values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the DELTA-H'degrees of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.