A SYNTHETIC PEPTIDIC SUBSTRATE OF MINIMAL SIZE AND SEMIOPTIMAL SEQUENCE FOR THE PROTEIN-TYROSINE KINASE PP60(C-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 t he protein tyrosine kinase pp60(c-src). The preferred amino acids surr ounding tyrosine were determined by a systematic study in which we inc reased the length of a series of linear peptides starting from the tri peptide EYG. Using an iterative cycle, the size of the peptide was inc reased 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 tripep tide EYG was not a substrate nor an efficient inhibitor. With increasi ng size of the peptide, the K-i decreased from 10.0 to 0.10 mM. The sm allest peptide to serve as a substrate was a hexapeptide. The best ove rall peptide obtained from this method, EFEYAFF, had a K-i value of 0. 13 mM with K-m and V-max values of 0.21 mM and 680 nmol/min/mg, respec tively. Our best peptide was found to have higher substrate specificit y than all other commerically available peptidic substrates for pp60(c -src). (C) 1996 Academic Press, Inc.