Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.
In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.
Suiko
M
M
Department of Biological Resource Sciences, Miyazaki University, Japan.
Fernando
P H
PH
Sakakibara
Y
Y
Nakajima
H
H
Liu
M C
MC
Abe
S
S
Nakatsu
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004926:Escherichia coli; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism; D013464:Sulfuric Acids / Q000737:chemistry; D014443:Tyrosine / Q000378:metabolism*
D006629:Hirudins; D011994:Recombinant Proteins; D013464:Sulfuric Acids; D014443:Tyrosine; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase