title: Cyclic peptides as probes of the substrate binding site of the cytosolic tyrosine kinase, pp60c-src.
abstract: A series of 48 cyclic peptides based on the amino acid sequence surrounding the autophosphorylation site of pp60(c-src) was synthesized and each was tested as both a substrate and an inhibitor of this protein tyrosine kinase. Starting with cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Ala6-Arg7-Gln8-d- Phe9-Pro10) a six-amino-acid survey was performed at positions 1 through 8 to determine which positions were critical for affinity and phosphorylation and which amino acids produced the greatest activity. Our survey found that Arg7 was detrimental for binding and phosphorylation and that aromatic residues were preferred at this position. Further increases in affinity were obtained with hydrophobic residues at position 6 with the optimum for both affinity and phosphorylation being Phe. Changes on the "amino-terminal" side of Tyr4 resulted in reduced Vmax values, illustrating the requirement for acidic residues in peptidic tyrosine kinase substrates. The result of the survey was cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Phe6-Phe7-Gln8-d-Phe 9-Pro10). The change of residues 6 and 7 resulted in a 42-fold increase in affinity and no increase in Vmax. As a substrate, this peptide displayed Michaelis-Menten kinetics at saturating ATP conditions. As an inhibitor, mixed inhibition was observed. A linear version of this peptide was 13-fold less potent an inhibitor than the cyclic peptide.
authors.affiliation: Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, 77030, USA. jsmcmur@audumla.mdacc.tmc.edu
authors.forename: J S
authors.initials: JS
authors.lastname: McMurray
authors.forename: R J
authors.initials: RJ
authors.lastname: Budde
authors.forename: S
authors.initials: S
authors.lastname: Ke
authors.forename: N U
authors.initials: NU
authors.lastname: Obeyesekere
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: Ramdas
authors.forename: C A
authors.initials: CA
authors.lastname: Lewis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D066298:In Vitro Techniques; D007700:Kinetics; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015335:Molecular Probes; D010455:Peptides; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D016392:Proto-Oncogene Proteins pp60(c-src)