Abstract
The kinetics of the forward tyrosyl protein sulfotransferase (TPS) reaction were examined using an assay based on the 35SO 4 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 K M value determined for BocCCK-8(ns) was 160 ± 18 μM, and that for [ 35S]PAPS was 0.15 ± 0.03 μM. 3′-Phosphoadenosine 5′-phosphate (3′-PAP) was found to be a product inhibitor with a Ki = 0.30 ± 0.02 μM. 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 μM. ATP inhibited competitively the reaction when PAPS was the varied substrate with a Ki = 3.6 ± 0.5 μM. 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.
Published Version
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