Analysis of the substrate specificity of tyrosylprotein sulfotransferase using synthetic peptides.
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.
Niehrs
C
C
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Kraft
M
M
Lee
R W
RW
Huttner
W B
WB
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
D009842:Oligopeptides; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase