Effect of Zn2+ during reactivation and refolding of urea-denatured creatine kinase

Abstract

The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn2+ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the "molten globule" state in the CK folding pathway. During refolding in the presence of Zn2+, creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.