Creatine kinase: essential arginine residues at the nucleotide binding site identified by chemical modification and high-resolution tandem mass spectrometry
| Autor: | Fred W. McLafferty, Troy D. Wood, Ziqiang Guan, Charles L. Borders, Lorenzo H. Chen, George L. Kenyon |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Phenylglyoxal
Arginine Tandem mass spectrometry Mass Spectrometry chemistry.chemical_compound Animals Nucleotide Binding site Muscle Skeletal Creatine Kinase chemistry.chemical_classification Multidisciplinary Binding Sites biology Nucleotides Active site Chemical modification Molecular biology Biochemistry chemistry Physical Sciences biology.protein Creatine kinase Rabbits Energy Metabolism |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America. 95(7) |
| ISSN: | 0027-8424 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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