Creatine kinase: Essential arginine residues at the nucleotide binding site identified by chemical modification and high-resolution tandem mass spectrometry

Creatine kinase: Essential arginine residues at the nucleotide binding site identified by chemical modification and high-resolution tandem mass spectrometry

^*Department of Chemistry, Natural Sciences Complex, State University of New York, Buffalo, NY 14260-3000; ^†Department of Chemistry, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263; ^‡Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301; ^§Department of Chemistry, College of Wooster, Wooster, OH 44691; and ^¶Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143

Contributed by Fred W. McLafferty

Abstract

Phenylglyoxal is an arginine-specific reagent that inactivates creatine kinase (CK). Previous results suggest that modification of the dimeric enzyme at a single arginine residue per subunit causes complete inactivation accompanied by the loss of nucleotide binding; the actual site of modification was not identified. Here, high-resolution tandem mass spectrometry (MS/MS) was used to identify three phenylglyoxal-modified Arg residues in monomeric rabbit muscle CK. Electrospray ionizaton Fourier-transform MS of the phenylglyoxal-modified CK that had lost ≈80% activity identified three species: unmodified, once-modified (+116 Da), and twice-modified (+232 Da) enzyme in a ratio of approximately 1:4:1. MS/MS restricts the derivatized sites to P122-P212 and P283-V332, whereas MS of Lys-C digestions revealed two modified peptides, A266-K297 and G116-K137. The only Arg in A266-K297 is Arg-291 (invariant), whereas MS/MS of modified G116-K137 shows that two of the three sites Arg-129, Arg-131, or Arg-134 (all invariant) can contain the modification. The recently reported x-ray crystal structure for the octameric chicken mitochondrial CK indicates that its nucleotide triphosphate-binding site indeed contains the equivalent of R291, R129, and R131 reported here to be at the active site of rabbit muscle CK.

Footnotes

↵ ‖ To whom correspondence should be addressed.
↵ ** The relative abundances of different charge states of the same ion are a function of the ion’s structure, so that the ratio of an ion’s abundance to that of its derivatized counterpart can vary with charge value.
ABBREVIATIONS:

CK,

creatine kinase;

ESI,

electrospray ionization;

MS,

mass spectrometry;

FT,

Fourier transform;

MS/MS,

tandem mass spectrometry;

NS,

nozzle/skimmer dissociation