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

ABSTRACT: 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 (Cys²⁸³) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys²⁸³ 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 Cys²⁸³ 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 Cys²⁸³ but was conclusive for the formation of Met⁷⁰-HETE and Met¹⁷⁹-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met⁷⁰Ala, Met¹⁷⁹Leu, and Met⁷⁰Ala/Met¹⁷⁹Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met⁷⁰ and Met¹⁷⁹ residues for CK activity.

SUBMITTER: Steinritz D

PROVIDER: S-EPMC8448711 | biostudies-literature |

REPOSITORIES: biostudies-literature

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