Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue

Protein S-thiolation, the formation of mixed disulphides of cysteine residu es in proteins with low-molecular-mass thiols, occurs under conditions asso ciated with oxidative stress and can lead to modification of protein functi on. In the present study, we examined the site of S-thiolation of the enzym e creatine kinase (CK), an important source of ATP in myocytes. Inactivatio n of this enzyme is thought to play a critical role in cardiac injury durin g oxidative stress, such as during reperfusion injury. Reaction of rabbit C K M isoenzyme with GSSG, used to model protein S-thiolation, was found to r esult in enzyme inactivation that could be reversed by GSH or dithiothreito l. Measurement of GSH that is released during the thiolation reaction indic ated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordi ngly, matrix-assisted laser-desorption ionization MS confirmed that the mol ecular mass of CK was increased, consistent with addition of one GSH molecu le/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the activ e-site cysteine residue (Cys(283)) was identified as the site of thiolation . Reversal of thiolation was shown to be rapid when GSH is abundant, render ing dethiolation of CK thermodynamically favoured within the cell. We concl ude that S-glutathionylation of CK could be one mechanism to explain tempor ary reversible loss in activity of CK during ischaemic injury. The maintain ance of GSH levels represents an important mechanism for regeneration of ac tive CK from S-glutathionylated CK.