A synthetic peptidic substrate of minimal size and semioptimal sequence for the protein tyrosine kinase pp60c-src.
We used a novel approach to determine the minimal size and semioptimal sequence of a peptide to serve as an inhibitor and/or substrate for the protein tyrosine kinase pp60c-src. The preferred amino acids surrounding tyrosine were determined by a systematic study in which we increased the length of a series of linear peptides starting from the tripeptide EYG. Using an iterative cycle, the size of the peptide was increased one residue at a time, first at the amino terminus and then at the carboxy terminus. A series of six analogs were synthesized at each position and assayed as inhibitors and substrates. The amino acids G, A, L, F, E, and K were used to semioptimize each position. The tripeptide EYG was not a substrate nor an efficient inhibitor. With increasing size of the peptide, the Ki decreased from 10.0 to 0.10 mM. The smallest peptide to serve as a substrate was a hexapeptide. The best overall peptide obtained from this method, EFEYAFF, had a Ki value of 0.13 mM with Km and Vmax values of 0.21 mM and 680 nmol/min/mg, respectively. Our best peptide was found to have higher substrate specificity than all other commerically available peptidic substrates for pp60c-src.
Ramdas
L
L
Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Obeyesekere
N U
NU
McMurray
J S
JS
Budde
R J
RJ
D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D017421:Sequence Analysis; D013379:Substrate Specificity; D019061:src-Family Kinases
D010455:Peptides; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C000631346:CSK protein, human