CYCLIC-PEPTIDES AS PROBES OF THE SUBSTRATE-BINDING SITE OF THE CYTOSOLIC TYROSINE KINASE, PP60(C-SRC)

A series of 48 cyclic peptides based on the amino acid sequence surrou nding the autophosphorylation site of pp60(c-src) was synthesized and each was tested as both a substrate and an inhibitor of this protein t yrosine kinase. Starting with cyclo(Asp(1)-Asn(2) -Gln(3-) r(4)-Ala(5) -Ala(6)-Arg(7)-Gln(8)-D-Phe(9)-Pro(10)) a six-amino-acid survey was pe rformed at positions 1 through 8 to determine which positions were cri tical for affinity and phosphorylation and which amino acids produced the greatest activity. Our survey found that Arg(7) was detrimental fo r binding and phosphorylation and that aromatic residues were preferre d at this position. Further increases in affinity were obtained with h ydrophobic residues at position 6 with the optimum for both affinity a nd phosphorylation being Phe. Changes on the ''amino-terminal'' side o f Tyr(4) resulted in reduced V-max values, illustrating the requiremen t for acidic residues in peptidic tyrosine kinase substrates. The resu lt of the survey was (Asp(1)-Asn(2)-Gln(3)-Tyr(4)-Ala(5)-Phe(6)-Phe(7) - Gln(8)-D-Phe(9)-Pro(10)). The change of residues 6 and 7 resulted in a 42-fold increase in affinity and no increase in V-max. As a substra te, 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. (C) 1998 Academic Press.