Catalytic mechanism of Golgi-resident human tyrosylprotein sulfotransferase-2: a mass spectrometry approach.
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.
Danan
Lieza M
LM
Department of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
Yu
Zhihao
Z
Ludden
Peter J
PJ
Jia
Weitao
W
Moore
Kevin L
KL
Leary
Julie A
JA
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*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D015238:Sulfotransferases; C114740:TPST2 protein, human
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