Cholecystokinin activation: evidence for an ordered reaction mechanism for the tyrosyl protein sulfotransferase responsible for the peptide sulfation.
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.
Frerot
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Laboratoire de Physiologie, Université René Descartes, Paris, France.
Vargas
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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
D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; C072106:desulfated benzyloxycarbonyl cholecystokinin (26-33); D010724:Phosphoadenosine Phosphosulfate; D002766:Cholecystokinin; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase