Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys(283)) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys(283) is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys(283) and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys(283) but was conclusive for the formation of Met(70)-HETE and Met(179)-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met(70)Ala, Met(179)Leu, and Met(70)Ala/Met(179)Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met(70) and Met(179) residues for CK activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00204-021-03137-6.