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

Journal Article (Journal Article)

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 approximately 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.

Full Text

Duke Authors

Cited Authors

  • Wood, TD; Guan, Z; Borders, CL; Chen, LH; Kenyon, GL; McLafferty, FW

Published Date

  • March 31, 1998

Published In

Volume / Issue

  • 95 / 7

Start / End Page

  • 3362 - 3365

PubMed ID

  • 9520370

Pubmed Central ID

  • PMC19840

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.95.7.3362


  • eng

Conference Location

  • United States