CREATINE-KINASE - ESSENTIAL ARGININE RESIDUES AT THE NUCLEOTIDE-BINDING SITE IDENTIFIED BY CHEMICAL MODIFICATION AND HIGH-RESOLUTION TANDEMMASS-SPECTROMETRY

Phenylglyoxal is an arginine-specific reagent that inactivates creatin e kinase (CK). Previous results suggest that modification of the dimer ic enzyme at a single arginine residue per subunit causes complete ina ctivation accompanied by the loss of nucleotide binding; the actual si te of modification was not identified, Here, high-resolution tandem ma ss spectrometry (MS/MS) was used to identify three phenylglyoxal-modif ied Arg residues in monomeric rabbit muscle CK, Electrospray ionizaton Fourier-transform MS of the phenylglyoxal-modified CK that had lost a pproximate to 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-P21 2 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-29 1 (invariant), whereas MS/MS of modified G116-K137 shows that two of t he three sites Arg-129, Arg-131, or Arg-134 (all invariant) can contai n the modification, The recently reported x-ray crystal structure for the octameric chicken mitochondrial CK indicates that its nucleotide t riphosphate-binding site indeed contains the equivalent of R291, R129, and R131 reported here to be at tile active site of rabbit muscle CK.