Hydrolysis of Tyrosine Sulfate in Acidic Solutions and the Occurence of Tyrosine Sulfation in the G Protein-Coupled Receptor PAC1

Dorte Balsved

Student thesis: Termpaper


#Tyrosine sulfation is a post-translational modification of proteins and peptides, which takes place in the trans-Golgi network and is catalyzed by two membrane-bound enzymes TPST-1 and TPST-2. In recent years several G protein-couples receptors (GPCRs) have been found to contain tyrosine sulfate. In this study the Pituitary Adenylate Cyclase Activating Polypeptide type 1 (PAC1) receptor was investigated, since the receptor contains four tyrosine sites in the N-terminal extracellular region that might be targets for sulfation. The four tyrosines were mutated through site-directed mutagenesis and the effect of these receptor mutations on ligand binding and signal transduction were determined. The results showed an approximately 3-fold increase in PAC1 receptor binding affinity towards the natural ligand PACAP-27, when the four tyrosines were mutated to phenylalanines, compared to the wild-type receptor. The same was observed in the signal transduction where the mutant receptor showed an approximately 3-fold increase in cAMP levels in response to PACAP-27 stimulation, indicating a role of these tyrosines in ligand binding or in correct receptor folding. Identification of sulfated tyrosines by purification of the recombinant receptor protein followed by mass spectrometry analysis was unsuccessful and evidence for tyrosine sulfation could not be determined. Sulfated tyrosines are not identified during Edmann sequencing of peptides and proteins, because the sulfate ester is very acid labile and rapidly hydrolyses to tyrosine in strong acidic solutions (pH <1). Little is known about the hydrolysis at mildly acidic solutions, which are used in several protein purification and analysis procedures (pH 1 to 4). In the present study sulfated gastrin-17 was used as a model tyrosine sulfated peptide, where the acid lability was investigated in the pH range 1 to 3 and at different temperatures, incubation times and with different acids. The results indicated that the tyrosine sulfate was fairly stable at the pH range 1 to 3 at room temperature and lower, only showing marginal hydrolysis. The acid hydrolysis was a pseudo first-order reaction and the rate constants measured at different temperatures followed the Arrhenius equation. The Arrhenius A factor of the hydrolysis reaction was determined which was within the range observed for unimolecular reactions. In addition the activation energy (Ea) of the reaction was calculated and showed a weakened S-O bond between the sulfuryl group and the phenol. The acid hydrolysis of sulfated gastrin-17 was compared to the hydrolysis of two other tyrosine sulfated peptides, drosulfokinin and caerulein, and it was shown that hydrolysis rate depended on the primary amino acid composition of the protein/peptide.

EducationsMolecular Biology, (Bachelor/Graduate Programme) Undergraduate or graduateChemistry, (Bachelor/Graduate Programme) Undergraduate or graduate
Publication date1 Jun 2006
SupervisorsPoul Erik Hansen


  • FPLC
  • Pituitary adenylate cyclase activating polypeptide type 1 receptor
  • Acid hydrolysis
  • Tyrosine sulfation
  • GPCR
  • PAC1