Creatine kinase: essential arginine residues at the nucleotide binding site identified by chemical modification and high-resolution tandem mass spectrometry.
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.
Wood
T D
TD
Department of Chemistry, Natural Sciences Complex, State University of New York, Buffalo, NY 14260-3000, USA.
Guan
Z
Z
Borders
C L
CL
Chen
L H
LH
Kenyon
G L
GL
McLafferty
F W
FW
D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism; D004734:Energy Metabolism; D013058:Mass Spectrometry; D018482:Muscle, Skeletal / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits
D009711:Nucleotides; D001120:Arginine; D003402:Creatine Kinase