Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = sigma ATP sigma creatine divided by (sigma ADP sigma creatine-P H+) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free Mg2+. The value was found to be 3.78 x 10(8) M-1 at free Mg2+ = 0 and 1.66 x 10(9) M-1 at free Mg2+ = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCKH+ = sigma ATP sigma creatine divided by (sigma ADP sigma creatine-P was 37.8 at free Mg2+ = 0 and 166 at free Mg2+ = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free Mg2+, being 0.391 at free Mg2+ = 0 and 1.05 at free Mg2+ = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free Mg2+, being 110 M at both free Mg2+ = 0 and free Mg2+ = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free Mg2+, being 272 M at free Mg2+ = 0 and 174 M at free Mg2+ = 0.89 x 10(-3) M.