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

Dirk Steinritz,Harald John,Markus Siegert,Tanja Popp,Horst Thiermann,Robin Lüling,Thomas Gudermann,Harald Mückter,Peter Reinemer

doi:10.1007/s00204-021-03137-6

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.

Highlights

Creatine kinase (CK, EC 2.7.3.2) belongs to an evolutionarily conserved group of enzymes
Alignment of rabbit muscle CK (rmCK) and hmCK using the Clustal Omega program (Sievers et al 2011) revealed an overall identity of the amino acid sequences of 96.6% with a complete matching of all Cys and Met positions. Cys283 was recently identified as a target of alkylating agents including sulfur mustard (SM) (Lüling et al 2021; Steinritz et al 2021)
As C ys283 in the active site of CK (Wang et al 2006) is supposed to bind creatine by electrostatic interactions between the free thiol-moiety of its side chain and the guanidine-group of creatine (Bong et al 2008), we assumed that a loss of CK activity is due to the alkylation of this residue after treatment with SM

Summary

Introduction

Creatine kinase (CK, EC 2.7.3.2) belongs to an evolutionarily conserved group of enzymes It catalyzes the reversible, magnesia-catalyzed (Mg) reaction between creatine (Cr) and adenosine triphosphate (ATP) forming phosphocreatine (PCr) and adenosine diphosphate (ADP) according to the reaction MgATP + Cr ⇆ MgADP + PCr (Schlattner et al 2016), being a key player in maintaining cellular energy homeostasis. The highly reactive Cys283 residue (amino acid numbering does include the N-terminal methionine (Met) residue) in the active site of the enzyme is discussed to play a pivotal role in this context (Maggio and Kenyon 1977; Bickerstaff and Price 1978; Furter et al 1993; Reddy et al 2000; Wang et al 2006) and is subject of current research

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