Kinetics and mechanism of the rat brain phenol sulphotransferase reaction.
Some properties of rat brain phenol sulphotransferase were investigated in in vitro at pH7.4. The enzyme was purified 10-fold by chromatography on DEAE-Sephadex -50. It can be assayed with 4-hydroxy-3-methoxyphenylethylene glycol or 4-methylumbelliferone as the sulphate acceptor. The partially purified enzyme is stable for at least 1 week when stored at 4 degrees C. It is, however, additionally activated (10--20%) and stabilized by 1 mM-dithiothreitol. The activity of the enzyme does not depend on the addition of exogenous Mg2+. The pH optima for the sulphation of 4-hydroxy-3-methoxyphenylethylene glycol and 4-methylumbelliferone are 7.8 and 7.4 respectively. Substrate inhibition by the sulphate acceptor is apparent at concentrations over 0.05mM. Initial-velocity studies in the absence and presence of product and dead-end inhibitors suggested that the mechanism of the rat brain sulphotransferase reaction is sequential ordered Bi Bi with a dead-end complex of enzyme with adenosine 3',5'-biphosphate and sulphate acceptor. The sulphate donor adenosine 3'-phosphate 5'-sulphatophosphate is the first substrate that adds to the enzyme, and the sulphate acceptor is the second substrate. The dissociation constant for the complex of enzyme with sulphate donor is 21 micron. The sulphated substrate is the first product and adenosine 3',5'-biphosphate is the second product that leaves the enzyme.
D000227:Adenine Nucleotides / Q000378:metabolism; D000249:Adenosine Monophosphate; D000818:Animals; D001921:Brain / Q000201:enzymology*; D004229:Dithiothreitol; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008734:Methoxyhydroxyphenylglycol / Q000378:metabolism; D010636:Phenols; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D051381:Rats; D013466:Sulfurtransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000227:Adenine Nucleotides; D010636:Phenols; D000249:Adenosine Monophosphate; D010724:Phosphoadenosine Phosphosulfate; D008734:Methoxyhydroxyphenylglycol; D013466:Sulfurtransferases; D008274:Magnesium; D004229:Dithiothreitol