title: Analysis of the substrate specificity of tyrosylprotein sulfotransferase using synthetic peptides.
abstract: Tyrosylprotein sulfotransferase (TPST) catalyzes the sulfation of proteins at tyrosine residues. We have analyzed the substrate specificity of TPST from bovine adrenal medulla with a novel assay, using synthetic peptides as substrates. The peptides were modeled after the known, or putative, tyrosine sulfation sites of the cholecystokinin precursor, chromogranin B (secretogranin I) and vitronectin, as well as the tyrosine phosphorylation sites of alpha-tubulin and pp60src. Varying the sequence of these peptides, we found that (i) the apparent Km of peptides with multiple tyrosine sulfation sites decreased exponentially with the number of sites; (ii) acidic amino acids were the major determinant for tyrosine sulfation, acidic amino acids adjacent to the tyrosine being more important than distant ones; (iii) a carboxyl terminally located tyrosine residue may be sulfated. Moreover, TPST catalyzed the sulfation of a peptide corresponding to the tyrosine autophosphorylation site of pp60v-src (Tyr-416) but not of a peptide corresponding to the non-autophosphorylation site of pp60c-src (Tyr-527). These results experimentally define structural determinants for the substrate specificity of TPST and show that this enzyme and certain autophosphorylating tyrosine kinases have overlapping substrate specificities in vitro.
authors.affiliation: Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
authors.forename: C
authors.initials: C
authors.lastname: Niehrs
authors.forename: M
authors.initials: M
authors.lastname: Kraft
authors.forename: R W
authors.initials: RW
authors.lastname: Lee
authors.forename: W B
authors.initials: WB
authors.lastname: Huttner
mesh_terms: D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism*; D013462:Sulfur Radioisotopes
chemical_list: D009842:Oligopeptides; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase