Sulfotransferases, PAPS

Sulfatation Alcohols or phenols Amines Sulfotransferases (PAPS) Sulfate esters Sulfonamides... 

The donor of the sulfate group in the compounds just mentioned i.s 3-phos-phoadenosine-5 -phosphDsuIfatie (PAPS). The structure of PAPS and its synthesis from ATP and free sulfate appear in Figure 10.54, FAI is used as a substrate by sulfotransferases. PAPS is synthesized in the cytoplasm and then transported into the Golgi, where it participates in the sulfation of maeromolcculcs destined for secretion from the cell. 

PAPS is synthesized within the cytosol of cells. Sulfate enters cells through an active transport process [4]. A sulfate transporter has been identified which is mutated in diastrophic dysplasia, a disease characterized by undersulfation of proteoglycans in cartilage [9]. For utilization by Golgi sulfotransferases, PAPS must cross a lipid bilayer. A PAPS translocase, which functions through an antiport mechanism has been characterized at the biochemical level [10]. 

Sulfotransferases catalyze the transfer of sulfate from PAPS to wide-range xenobiotics that possess hydroxyl groups. Steroid alcohols are among the endogenous substrates. The sulfotransferases exist in different forms. 

The role of N-sulfonyloxy arylamines as ultimate carcinogens appears to be limited. For N-hydroxy-2-naphthylamine, conversion by rat hepatic sulfotransferase to a N-sulfonyloxy metabolite results primarily in decomposition to 2-amino-l-naphthol and 1-sulfonyloxy-2-naphthylamine which are also major urinary metabolites and reaction with added nucleophiles is very low, which suggests an overall detoxification process (9,17). However, for 4-aminoazobenzene and N-hydroxy-AAF, which are potent hepatocarcinogens in the newborn mouse, evidence has been presented that strongly implicates their N-sulfonyloxy arylamine esters as ultimate hepatocarcinogens in this species (10,104). This includes the inhibition of arylamine-DNA adduct formation and tumorigenesis by the sulfotransferase inhibitor pentachlorophenol, the reduced tumor incidence in brachymorphic mice that are deficient in PAPS biosynthesis (10,115), and the relatively low O-acetyltransferase activity of mouse liver for N-hydroxy-4-aminoazobenzene and N-OH-AF (7,114,115). 

The sulfotransferases catalyze formation of sulfate esters of compounds having hydroxy or amino groups. The donor in this transfer of a sulfuryl group is 3 -phosphoadenylsulfate, also named previously as 3 -phosphoadenosine-5 -phos-phosulfate (PAPS). An example of this reaction is seen in Eq. (11) for phenol as the sulfuryl (S03) acceptor, with the products of the reaction being phenyl sulfate and adenosine 3, 5 -bisphosphate (PAP). 

Conjugations can also be brought about by sulfotransferases (SULTs) and glutathi-one-S-transferases (GSTs), both of which exist in a number of isoenzymic forms. Amines and alcohols are sulfate acceptors and SULTs are important in steroid hormone and catecholamine metabolism and like the UGTs require the sulfate to be activated prior to its incorporation into the target molecule (Figure 6.32). In this case, sulfate is activated at the expense of two molecules of ATP to form the final sulfate carrier PAPS O -phosphoadenosine-S -phosphosulfate). 

Protein sulfation occurs exclusively at tyrosine residues." It has been suggested that up to 1 % of the tyrosine protein content becomes sulfated, which is the most abundant posttranslational modification for tyrosine, with phosphorylation occurring only on 0.5% of tyrosine protein content." Sulfation occurs mostly on excreted proteins or trans-membrane proteins. Sulfation is catalyzed by tyrosylprotein sulfotransferase (TPST), with PAPS as a cosubstrate (Scheme 4). Like kinases, sulfotransferases have a biological inverse known as sulfatases." ... 

Sulfotransferases (SULTs) are important for the metabolism of a number of drugs, neurotransmitters, and hormones, especially the steroid hormones. The cosubstrate for these reactions is 3 -phosphoadenosine 5 -phosphosulfate (PAPS) (Fig. 4.1). Like the aforementioned enzymes, sulfate conjugation typically renders the compound inactive and more water soluble. However, this process can also result in the activation of certain compounds, such as the antihypertensive minoxidil and several of the steroid hormones. Seven SULT isoforms identified in humans, including SULTs lAl to 1A3, possess activity toward phenolic substrates such as dopamine, estradiol, and acetaminophen. SULTIBI possesses activity toward such endogenous substrates as dopamine and triiodothyronine. SULTIEI has substantial activity toward steroid hormones, especially estradiol and dehydroepiandrosterone, and toward the anti-... 

Sulfation of the carbohydrate chain occurs after the monosaccharide to be sulfated has been incorporated into the growing carbohydrate j chain. The source of the sulfate is 3 -phosphoadenosyl-5 -phospho-sulfate (PAPS, a molecule of AMP with a sulfate group attached to the 5-phosphate). Sulfotransferases cause the sulfation of the carbohydrate chain at specific sites. [Note An example of the synthesis of a sulfated glycosaminoglycan, chondroitin sulfate, is shown in Figure 14.11.] PAPS is also the sulfur donor in glycosphingolipid synthesis. [Note A defect in the sulfation process results in one of several autosomal recessive disorders that affect the proper development and maintenance of the skeletal system. This illustrates Ihe importance of the sulfation step.]... 

Sulfotransferase activity is not restricted to minoxidil. The ability of other pyrimidine-, as well as pyridine-, triazine- and imidazole N-oxides to serve as substrates was investigated using soluble liver preparation and PAPS. The variety of structures studied indicated that heteroaromatic N-oxides are generally metabolized by sulfotransferases183. Presumably, all of the heterocycles tested were conjugated via their N-oxide oxygens. 

Figure 4. Activities of cerebroside PAPS sulfotransferase (CST) monogalactosyl diacylglycerol PAPS sulfotransferase (MST) and hydroxy fatty acyl ceramide UDP-galactose galactosyltransferase (C Gal. T) in reconstituted homogenates of dissociated brain cells from 15-day embryonic mice grown for varying days in culture. Note that the activity of the galactosyltransferase is expressed on a different scale from that of the sulfotransferase (21).

Conditions for cytosolic incubations depend on the aim of the assay e.g. to cover specific enzymatic activity present in the cytosol. For this purpose it is essential to fortify the incubation medium with the specific cofactor for the reaction-if needed (Ekins 1999). (J> -Nicotinamide adenine dinucleotide (NAD) is needed for alcohol and aldehyde dehydrogenases, flavin adenine dinucleotide (FAD) for polyamine oxidase, P-nicotinamide adenine dinucleotide phosphate (NADPH) for Dihydropyrimidine dehydrogenase. Phase II reactions depend on PAPS (sulfotransferases,... 

Chen et al. (1999,2003) used cytosol prepared from various sections of the human intestine to study the occurrence and distribution of sulfotransferases in the gastrointestinal tract. They fortified the cytosol with PAPS. They utilized the sulfuryl group transfer from p-nitrophenol sulphate to PAP to generate PAPS for measurement of the phenol sulfotransferase activity by measurement of the colored product p-nitro-phenol. Cytosolic incubation were stopped by addition of Tris buffer, pH 8.7. 

Sulfotransferases (SULTs) are cytosolic phase II detoxification enzymes involved in sulfonation of various xenobiotics and endobiotics. There are also membrane-bound SULTs that are not involved in phase II metabolism but are involved in the sulfonation of proteins and polysaccharides. Substrates of cytosolic SULTs include alcohols (ethanol, 2-butanol, cholesterol, bile acids), phenols (phenol, naphthol, acetaminophen), aromatic amines and hydroxyamines (2-naphthylamine, A-hydroxy 2-naphthylamine). SULTs transfer sulfonate (S03) to a hydroxy or amino group of a substrates from the cofactor 3 -phosphoadenosine-5 -phosphosulfate (PAPS), generating highly water-soluble metabolites for elimination through the kidney and liver. 

Kinases and sulfotransferases utilize similar substrates and catalyze similar reactions. Both transfer anionic groups (Scheme 14.9). Both enzyme classes are capable of binding adenosine-based substrates. Sulfotransferases bind 3 -phospho-adenosine-5 -phosphosulfate (PAPS) (35) as a sulfate donor and kinases bind adenosine-5 -triphosphate (ATP) (36) as a phosphoryl donor. 

Estrogen sulfotransferase green with consumed cofactor (PAP) and substrate (17(3-estradiol) in yellow uridylate kinase blue with consumed cofactor (ADP) and substrate analog (ADP) in red. 

PAPS-competitive NodH inhibitors (25, Scheme 14.7) with modest inhibitory activity (IC50 values ranging from 20 to 40 pM) were found that showed selectivity among several tested sulfotransferases. They all displayed inhibitory activity in the micromolar range against several kinases [62],... 

Figure 9,19 Chromatograms of the cosubstrate (PAPS) and products of liver phenol sulfotransferase activity. Upper tracing a, PAPS, b, sulfate-conjugated dopamine. Middle tracing a, PAPS, b, sufate-conjugated Dopac. Lower tracing a, PAPS, b, sulfate-conjugated phenol. The retention time for sulfate-conjugated dopamine, Dopac, and phenol was 4.5, 6.8, and 7.0 minutes, respectively. (From Sim and Hsu, 1990.)...

Sulfotransferase catalyzes the transfer of sulfate from the donor molecule adenosine-3 -phosphate-5 -phosphosulfate (PAPS) to an acceptor, /3-naphthol, to form the reaction product /3-naphthol sulfate. 

Figure 9.146 HPLC analysis of PAP in an aryl sulfotransferase IV reaction mixture. The reaction mixture and incubation conditions were 50 fiM 1-naphthalenemethanol and 2.9 fig of AST IV. The mobile phase for HPLC analysis contained 12% methanol in 75 mM potassium phosphate (pH 5.45), 100 mM ammonium chloride, and 1.0 mM 1-octylamine. The flow rate was 2.0 mL/min, and detection was at 254 nm, with a full scale sensitivity of 0.02 AU. Sample injection is indicated by an arrow. (From Duffel et al., 1989.)...

Sulfation. Sulfotransferases use 3-phosphoadenosine-5-phosphosulfate (PAPS) as a cosubstrate. It concerns mostly hydroxyl groups. 

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