Reactivation kinetics of guanidine hydrochloride-denatured creatine kinase measured using the substrate reaction

Guanidine hydrochloride-denatured creatine kinase (CK) can very quickly form a dimer with reactivity when the denaturant is diluted into the reaction system in the presence of DTT or EDTA. Tsou's method and its applied equation [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381–436; Yang and Zhou (1998), Biochim. Biophys. Acta 1388, 190–198] were used to measure the kinetic reactivation rate constants and the reactivation degree for reassociated CK dimers. Partial reactivation (about 50% at best) occurred following a monophasic course during the substrate reaction when compared with previous time interval measurements. The reactivation degree increased with increasing DTT (0.1–5 mM) and EDTA (0.1–1 mM) concentrations. The apparent forward rate constants do not change with DTT concentration, showing that the reactivation is a reversible first-order reaction, but not of complex formation type. However, the apparent forward rate constants do change with EDTA concentration, showing that the reactivation with EDTA is a reversible first-order reaction as well as of complex formation type. Excess DTT concentrations have an inhibitory effect, indicating that the excessive EDTA acts as a metal chealate not only for free Mg²⁺, but also for MgATP during the enzyme catalysis. This study shows that additional information about the reactivation of CK can be obtained from examining the substrate reaction. The possible refolding pathway of CK is discussed.