Free radical induced inactivation of creatine kinase: sites of interaction, protection, and recovery

The study aims at a clarification of the oxidative damage of creatine kinase isoenzymes by X-ray-induced water radiolysis. The radical species generated by this method (under appropriate conditions) are similar to those discussed in the context of mitochondrial energy metabolism. The decay of the enzyme activity is accompanied by a strong decrease of the number of accessible SH groups and by a reduction of the endogenous tryptophan fluorescence. Free radical effects are diminished if irradiation is carried out in the presence of 2-mercaptoethanol. Partial recovery of the activity (repair) is observed if 2-mercaptoethanol is added after irradiation. The experiments suggest a twofold importance of thiol reagents (RSH): to reduce the concentration of free radicals by scavenger reactions and to modify the inactivation mechanism in such a way that efficient repair of enzyme damage may be achieved. Cysteine 282 of MM-CK (Cys-278 in the case of Mi-CK) seems to play a crucial role in this respect. Blockage of the SH group of cysteine 282 by oxidized glutathione effectively protects the enzyme against inactivation by NO(*)(2) radicals. In the absence of nitrogen dioxide and of thiol reagents, however, inactivation seems to proceed via a less specific mechanism involving additional targets of the enzyme.