THE PURIFICATION AND CHARACTERIZATION OF THE CATALYTIC DOMAIN OF SRC EXPRESSED IN SCHIZOSACCHAROMYCES-POMBE - COMPARISON OF UNPHOSPHORYLATED AND TYROSINE-PHOSPHORYLATED SPECIES

The catalytic domain of chicken Src including the C-terminal tail (Src -CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity, The expressed protein is a mixture of unphosphorylated (8 0%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric met hod that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at T yr416 or at Tyr436. The stability of Src-CD is comparable to the wild- type protein. Src-CD auto-phosphorylates and efficiently phosphorylate s substrate peptides and proteins. Auto-phosphorylation occurs by an i ntermolecular mechanism and is completely inhibited by an excess of su bstrate peptide. Kinetic measurements for two exogenous substrates, th e Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showe d that the overall activity (k(cat)) of the Src-CD molecule is about 1 0 times higher than that of wild-type Src. The k(cat) values for phosp horylation of the Src substrate peptide are similar for the unphosphor ylated and monophosphorylated Src-CD (50 min(-1)), but the apparent K- m values differ significantly (approximately 3 mu M and 10 mu M, respe ctively). Therefore, at low substrate concentrations in vitro the mono -phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K-m values of the mono-ph osphorylated Src-CD and wild-type Src for the Src substrate peptide an d enolase are similar, indicating that, under these conditions, the ki nase domain is mainly responsible For substrate binding.