Rapid and irreversible inhibition of creatine kinase by peroxynitrite

We examined the ability of peroxynitrite and other .NO-derived oxidants to inhibit creatine kinase (CK). Peroxynitrite potently inhibited CK activity and depleted protein thiols. The rate constant for this reaction was 8.85x10(5) M(-1) s(-1). Glutathione did not reactivate CK activity nor did it regenerate protein thiol content. In contrast, glutathione reactivated CK, and regenerated protein thiols, after inhibition by either .NO or oxidized glutathione (GSSG). Peroxynitrite did not irreversibly inhibit CK after it had been treated with GSSG to block protein thiols. We conclude that thiol oxidation is a critical event leading to inactivation of CK by peroxynitrite.