title: Cholecystokinin activation: evidence for an ordered reaction mechanism for the tyrosyl protein sulfotransferase responsible for the peptide sulfation.
abstract: The kinetics of the forward tyrosyl protein sulfotransferase (TPS) reaction were examined using an assay based on the 35SO4 transfer from 3'-phosphoadenosine 5'-phospho(35S)sulfate [( 35S]PAPS) to tyrosyl residues of the non-sulfated cholecystokinin derivative, BocCCK-8(ns). TPS present in the microsomal membranes from rat cerebral cortex was used for these studies. Initial velocity measurements performed over a wide range of PAPS, BocCCK-8(ns), 3'-PAP and BocCCK-8(s) concentrations, indicated that the reaction follows an ordered mechanistic pathway. The KM value determined for BocCCK-8(ns) was 160 +/- 18 microM, and that for [35S]PAPS was 0.15 +/- 0.03 microM. 3'-Phosphoadenosine 5'-phosphate (3'-PAP) was found to be a product inhibitor with a Ki = 0.30 +/- 0.02 microM. BocCCK-8(s) produced an uncompetitive inhibition pattern on the TPS reaction. Adenosine 5'-phosphosulfate (APS) behaved as a competitive inhibitor versus PAPS with a Ki = 3.0 +/- 0.3 microM. ATP inhibited competitively the reaction when PAPS was the varied substrate with a Ki = 3.6 +/- 0.5 microM. The results of product and substrate inhibition studies and the patterns of dead end inhibition obtained with APS are best fit by an ordered Bi-Bi reaction mechanism where PAPS is the first substrate to bind and 3'-PAP is the last product to be released.
authors.affiliation: Laboratoire de Physiologie, Université René Descartes, Paris, France.
authors.forename: O
authors.initials: O
authors.lastname: Frerot
authors.forename: F
authors.initials: F
authors.lastname: Vargas
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002766:Cholecystokinin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008861:Microsomes / Q000201:enzymology*; D010446:Peptide Fragments / Q000494:pharmacology*; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D051381:Rats; D015238:Sulfotransferases / Q000378:metabolism*; D013462:Sulfur Radioisotopes
chemical_list: D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; C072106:desulfated benzyloxycarbonyl cholecystokinin (26-33); D010724:Phosphoadenosine Phosphosulfate; D002766:Cholecystokinin; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase