title: Reaction product inactivation of aryl sulfotransferase IV following electrophilic substitution by the sulfuric acid ester of N-hydroxy-2-acetylaminofluorene.
abstract: Rat liver N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) sulfotransferase activity is mediated by aryl sulfotransferase IV (AST IV) and causes the bioactivation of N-OH-2AAF to a highly reactive sulfuric acid ester form putatively capable of inducing liver cancer. Dietary administration of 2-acetylaminofluorene (2AAF) to induce hepatocarcinogenesis in rats has been shown to cause a rapid loss in N-OH-2AAF sulfotransferase activity. A possible mechanism for the in vivo loss in sulfotransferase activity may be the PAPS-dependent, sulfotransferase-catalyzed, reaction product inactivation of the enzyme by covalent reaction with the N-OH-2AAF sulfuric acid ester. In vitro studies to evaluate this possibility utilized a highly purified form of AST IV and measured the extent of PAPS-dependent interaction between the enzyme and N-OH-2[9-14C]AAF. The results showed the presence of a adenosine-3'-phospho-5'-phosphosulfate (PAPS)-dependent 14C-labeling of AST IV. The labeling could be blocked if the sulfotransferase inhibitor pentachlorophenol was present. Analysis of 14C-labeled AST IV following alkaline digestion and chromatography of digestion products indicated that AST IV cysteine and methionine residues were primary sites of 2[9-14C]AAF adduction. Studies involving the pretreatment of AST IV with PAPS and N-OH-2AAF prior to the measurement of N-OH-2AAF sulfotransferase activity showed a close parallel between formation of the AST IV cysteine-2AAF adduct and loss of activity. Similar studies showed that enzyme inactivation and cysteine-2AAF adduct formation could be blocked when excessive amounts of a competing nucleophile, methionine, were present during the pretreatment step, suggesting that inactivation does not proceed by a mechanism-based process. Finally, experiments involving prior reaction of AST IV with the thiol-blocking agent, N-ethylmaleimide, before measurement of enzyme activity showed essentially full loss of sulfotransferase activity and suggested that formation of AST IV cysteine-2AAF adducts could be a mechanism for enzyme inactivation. These results indicate that the in vitro inactivation of AST IV by the reactive N-OH-2AAF sulfuric acid ester is accompanied by covalent binding to AST IV, possibly through the formation of cysteine-2AAF adducts, and suggests that this mechanism merits further consideration as a basis for the loss of N-OH-2AAF sulfotransferase activity in vivo.
authors.affiliation: Samuel Roberts Noble Foundation, Inc., Biomedical Division, Ardmore, OK 73402.
authors.forename: D P
authors.initials: DP
authors.lastname: Ringer
authors.forename: T R
authors.initials: TR
authors.lastname: Norton
authors.forename: R R
authors.initials: RR
authors.lastname: Self
mesh_terms: D000818:Animals; D015239:Arylsulfotransferase / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004952:Esters; D005033:Ethylmaleimide / Q000494:pharmacology; D006881:Hydroxyacetylaminofluorene / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008715:Methionine / Q000378:metabolism; D051381:Rats
chemical_list: D004952:Esters; D006881:Hydroxyacetylaminofluorene; D008715:Methionine; D015239:Arylsulfotransferase; D003545:Cysteine; D005033:Ethylmaleimide