title: Catalytic mechanism of Golgi-resident human tyrosylprotein sulfotransferase-2: a mass spectrometry approach.
abstract: Human tyrosylprotein sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network, affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this study, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites, and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident sulfotransferase.
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
authors.forename: Lieza M
authors.initials: LM
authors.lastname: Danan
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Yu
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Ludden
authors.forename: Weitao
authors.initials: W
authors.lastname: Jia
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Moore
authors.forename: Julie A
authors.initials: JA
authors.lastname: Leary
mesh_terms: D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D006056:Golgi Apparatus / Q000737:chemistry / Q000201:enzymology*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015238:Sulfotransferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D015238:Sulfotransferases; C114740:TPST2 protein, human
references.citation: J Biochem. 2006 Nov;140(5):731-7
references.pmid: 17028309
references.citation: Chem Biol. 2000 Mar;7(3):R57-61
references.pmid: 10712936
references.citation: J Biol Chem. 1990 May 25;265(15):8525-32
references.pmid: 2341394
references.citation: Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2896-901
references.pmid: 9501187
references.citation: FASEB J. 1997 Mar;11(4):206-16
references.pmid: 9068609
references.citation: Toxicol Sci. 2006 Mar;90(1):5-22
references.pmid: 16322073
references.citation: Eur J Biochem. 1990 Mar 30;188(3):587-96
references.pmid: 2331987
references.citation: Biochemistry. 2008 Oct 28;47(43):11251-62
references.pmid: 18834145
references.citation: Arch Biochem Biophys. 1997 Jan 1;337(1):17-26
references.pmid: 8990263
references.citation: J Immunol. 2000 Nov 1;165(9):5295-303
references.pmid: 11046064
references.citation: Protein Sci. 2004 Apr;13(4):903-12
references.pmid: 15044725
references.citation: Eur J Biochem. 1990 Mar 30;188(3):577-86
references.pmid: 2331986
references.citation: Biochemistry. 1985 May 7;24(10):2477-82
references.pmid: 3860259
references.citation: Nat Struct Biol. 1997 Nov;4(11):904-8
references.pmid: 9360604
references.citation: Trends Biochem Sci. 1998 Apr;23(4):129-30
references.pmid: 9584614
references.citation: J Biol Chem. 2004 Oct 22;279(43):45185-93
references.pmid: 15304505
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11134-9
references.pmid: 9736702
references.citation: Chem Biol. 1999 Jan;6(1):R9-R22
references.pmid: 9889154
references.citation: EMBO J. 1990 Jan;9(1):35-42
references.pmid: 2295314
references.citation: Mol Endocrinol. 2007 Jul;21(7):1713-21
references.pmid: 17456791
references.citation: Arch Biochem Biophys. 1986 Sep;249(2):464-71
references.pmid: 3463246
references.citation: Mol Cell Biol. 2006 Aug;26(15):5838-49
references.pmid: 16847335
references.citation: Cell. 1999 Mar 5;96(5):667-76
references.pmid: 10089882
references.citation: FEBS Lett. 1998 Aug 21;433(3):211-4
references.pmid: 9744796
references.citation: Biochem Biophys Res Commun. 2002 Feb 1;290(4):1206-13
references.pmid: 11811991
references.citation: FASEB J. 1997 Jan;11(1):1-2
references.pmid: 9034159
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3497-500
references.pmid: 12824352
references.citation: Biochem Pharmacol. 2004 Apr 1;67(7):1355-61
references.pmid: 15013851
references.citation: J Biol Chem. 1994 Apr 8;269(14):10883-90
references.pmid: 8144675
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19217-22
references.pmid: 19036922
references.citation: J Biol Chem. 1985 May 25;260(10):6434-9
references.pmid: 3922974
references.citation: J Am Soc Mass Spectrom. 2008 Oct;19(10):1459-66
references.pmid: 18672380
references.citation: J Biol Chem. 1992 Jan 25;267(3):1858-63
references.pmid: 1309801
references.citation: Anal Biochem. 1990 Feb 1;184(2):274-8
references.pmid: 2327571
references.citation: Curr Opin Drug Discov Devel. 2000 Sep;3(5):502-15
references.pmid: 19649879
references.citation: J Biol Chem. 1969 Nov 10;244(21):5808-19
references.pmid: 5350937
references.citation: Biochem Biophys Res Commun. 2001 Jul 13;285(2):526-9
references.pmid: 11444874
references.citation: J Biol Chem. 1998 Oct 16;273(42):27325-30
references.pmid: 9765259
references.citation: Biochemistry. 1983 Sep 27;22(20):4637-41
references.pmid: 6626521
references.citation: J Biol Chem. 2004 Apr 9;279(15):14726-33
references.pmid: 14736884
references.citation: Biochim Biophys Acta. 2000 Apr 6;1474(2):115-27
references.pmid: 10742590
references.citation: Proc Natl Acad Sci U S A. 1985 Sep;82(18):6143-7
references.pmid: 3862121
references.citation: J Biol Chem. 2003 Jul 4;278(27):24243-6
references.pmid: 12730193
references.citation: J Biol Chem. 2002 Aug 16;277(33):29484-9
references.pmid: 12034737
references.citation: J Biol Chem. 2002 May 17;277(20):17928-32
references.pmid: 11884392
references.citation: Biochem Biophys Res Commun. 2009 May 22;383(1):83-7
references.pmid: 19344693
references.citation: Biochemistry. 2002 Nov 5;41(44):13283-8
references.pmid: 12403630
references.citation: Biochemistry. 2006 Apr 25;45(16):5122-8
references.pmid: 16618101
references.citation: J Biol Chem. 1998 May 1;273(18):10888-92
references.pmid: 9556564
references.citation: J Biol Chem. 2002 May 31;277(22):19418-23
references.pmid: 11909868
references.citation: Anal Biochem. 2005 Jun 1;341(1):94-104
references.pmid: 15866533
references.citation: Methods Enzymol. 1977;47:431-42
references.pmid: 22021
references.citation: J Biol Chem. 1998 Sep 18;273(38):24770-4
references.pmid: 9733778
references.citation: J Biol Chem. 2004 Jun 11;279(24):25789-97
references.pmid: 15060080
references.citation: J Biol Chem. 1998 Dec 25;273(52):35381-7
references.pmid: 9857081