Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK.
The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, +1 and +3. The synthetic peptides were tested as substrates for the p43v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.
Ruzza
P
P
CNR Centre for the Study of Biopolymers, Department of Organic Chemistry, University of Padua, Italy.
Calderan
A
A
Filippi
B
B
Donella-Deana
A
A
Pinna
L A
LA
Borin
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D016313:Genes, abl; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D009842:Oligopeptides; D011505:Protein-Tyrosine Kinases