EXPLORATION OF THE SEQUENCE SPECIFICITY OF PP60(C-SRC) TYROSINE KINASE - MINIMAL, PEPTIDE SEQUENCE REQUIRED FOR MAXIMAL ACTIVITY

The minimum length required for phosphorylation of a peptide by pp60(c-src) tyrosine kinase (srcTK) was delineated in this work, Budde (M. D, Anderson University of Texas, personal communication) suggested that the peptide (FGE)(3)Y(GEF)(2)GD (peptide I) was a ''good'' srcTK substrate. Peptide I yielded a 251-fold higher k(cat)/K-m than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK, This was due to a 38-fold lower K-m and a 6.6-fold increase in k(cat). 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 K-m. 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 k(cat)/K-m relative to peptide I. The latter was due primarily to an increase in K-m. 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.