The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

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  • Additional Information
    • Source:
      Publisher: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 0107600 Publication Model: Print Cited Medium: Print ISSN: 0014-2956 (Print) Linking ISSN: 00142956 NLM ISO Abbreviation: Eur J Biochem Subsets: MEDLINE
    • Publication Information:
      Publication: -2004: Oxford, UK : Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
      Original Publication: Berlin, New York, Springer.
    • Subject Terms:
      src Homology Domains*; Schizosaccharomyces/*genetics ; src-Family Kinases/*chemistry ; src-Family Kinases/*genetics; Amino Acid Sequence ; Animals ; Binding Sites/genetics ; Catalysis ; Chickens ; Enzyme Stability ; Kinetics ; Mass Spectrometry ; Molecular Sequence Data ; Peptide Mapping ; Peptides/chemistry ; Phosphorylation ; Substrate Specificity ; Tyrosine/chemistry ; src-Family Kinases/metabolism
    • Abstract:
      The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(m) values differ significantly (approximately 3 microM and 10 microM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.
    • Accession Number:
      0 (Peptides)
      42HK56048U (Tyrosine)
      EC 2.7.10.2 (src-Family Kinases)
    • Publication Date:
      Date Created: 19960915 Date Completed: 19961127 Latest Revision: 20190620
    • Publication Date:
      20231215
    • Accession Number:
      10.1111/j.1432-1033.1996.0756h.x
    • Accession Number:
      8856081