Journal of Lipid Research, Vol 27, 1111-1123, Copyright © 1986 by Lipid Research, Inc.
Evidence for an ordered reaction mechanism for bile salt: 3'phosphoadenosine-5'-phosphosulfate: sulfotransferase from rhesus monkey liver that catalyzes the sulfation of the hepatotoxin glycolithocholate
S Barnes, R Waldrop, J Crenshaw, RJ King and KB Taylor
The in vivo formation of the sulfate ester of glycolithocholate is a
critical step in the elimination of this hepatotoxic bile salt. Rhesus
monkeys fed chenodeoxycholate or ursodeoxycholate, the precursors of
lithocholate, develop frank cirrhosis in association with accumulation of
nonsulfated glycolithocholate in bile. An enzyme catalyzing the formation
of glycolithocholate-3-sulfate has been isolated from hepatic cytosol of
adult female rhesus monkeys and has been purified 146-fold. When reduced it
appears as a 30 kD band on an SDS-polyacrylamide gradient gel. It has a pH
optimum of 7.0 and is stimulated by low concentrations of Mg2+ (up to 2
mM), but does not have an absolute requirement for this metal ion. The
kinetics of this enzyme have been investigated to ascertain whether its
reaction mechanism can account for the poor in vivo rate of
glycolithocholate sulfation. Inhibitor studies with an oxidized metabolite
of lithocholate, 3-keto-5 beta- cholanoate, showed that the latter is a
competitive inhibitor of glycolithocholate and is noncompetitive with the
active form of sulfate, 3'phosphoadenosine-5'-phosphosulfate. The
monophosphonucleotide 3'-AMP is a competitive inhibitor of
3'phosphoadenosine-5'-phosphosulfate, and is noncompetitive with
glycolithocholate. These observations are consistent with a sequentially
ordered Bi Bi reaction mechanism in which the bile salt is the first
substrate to bind to the enzyme. Such a reaction mechanism for bile
salt:3'phosphoadenosine-5'-phosphosulfate:sulfotransferase would be,
therefore, the first time in which the sulfate acceptor (the bile salt) is
the initial substrate to bind to a sulfotransferase. These studies have
shown that although rhesus monkeys have a liver enzyme capable of forming
the sulfate ester of glycolithocholate, its reaction mechanism and the
potent inhibition caused by simple metabolites, such as 3-keto-5
beta-cholanoate, may serve to under- express the activity of the enzyme in
vivo.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
