Exploration of the sequence specificity of pp60c-src tyrosine kinase. Minimal peptide sequence required for maximal activity.
The minimum length required for phosphorylation of a peptide by pp60c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M.D. Anderson University of Texas, personal communication) suggested that the peptide (FGE)3Y(GEF)2GD (peptide I) was a "good" srcTK substrate. Peptide I yielded a 251-fold higher kcat/Km than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower Km and a 6.6-fold increase in kcat.N-terminal truncation of up to 8 residues in a series of peptides yielded only a 3-fold decrease in activity. Removal of the final N-terminal residue resulted in a 10-fold loss in substrate activity, primarily as a result of an increase in the Km. C-terminal truncations ending in the amide yielded no significant loss in activity until the Y + 3 residue was removed, which resulted in a 73-fold decrease in kcat/Km relative to peptide I. The latter was due primarily to an increase in Km. The results from peptides truncated on both termini suggest that subsite recognition N- and C-terminal relative to the site of phosphorylation can be examined independently. In addition, the observation that only 5 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interactions with the phosphoacceptor site may be developed.
Edison
A M
AM
Division of Molecular Sciences, Glaxo Research Institute, Research Triangle Park, North Carolina 27709, USA.
Barker
S C
SC
Kassel
D B
DB
Luther
M A
MA
Knight
W B
WB
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D013379:Substrate Specificity
D009842:Oligopeptides; D010455:Peptides; D016392:Proto-Oncogene Proteins pp60(c-src)