In vitro determination of creatine kinase substrate fluxes using 31P-nuclear magnetic resonance

Forward (kf) and reverse (kr) rate constants and the corresponding flux rates of the creatine kinase catalysed reaction between creatine phosphate (CrP) and adenosine triphosphate (ATP); CrP + ADP<-->kf kr ATP + Cr were measured in vitro at 295 K. Both rate constants were determined using magnetic resonance saturation transfer techniques. To study the dependence of kr and kf on the creatine kinase concentration, the creatine kinase activity was varied from 2400 to 75 U.ml-1. At equilibrium and high creatine kinase activities, the forward to reverse flux rate ratios are close to 1. A dispersion in the reaction rate constants was observed at activities < or = 600 U.ml-1. We measured kr > kf for all enzyme activities below 600 U.ml-1. This observation could partially be explained by the presence of ATPase contamination in the enzyme. These findings are relevant for the in vivo studies of creatine kinase activity in the presence of multi-site phosphate exchange in cellular ATP-pools. As mitochondrial creatine kinase is not in equilibrium these results are also of interest in this area.