Adenosine 5 -phosphosulfate from

FIGURE 6.3 Quantification on the first half of an isolated peak. The spectrum is from the [2Fe-2S] cluster in the enzyme adenosine phosphosulfate reductase from Desulfovibrio vulgaris (Verhagen et al. 1993). The inset shows the asymmetrical low-field -feature the vertical line at the peak position indicates the rightmost integration limit for quantification on half... 

FIGURE 13.6 Whole bacterial-cell EPR. A frozen concentrated suspension of cells from the sulfate-reducing bacterium Desulfovibrio vulgaris gives an EPR spectrum with only a [2Fe-2S]1+ signal and a flavin radical signal, both from adenosine phosphosulfate reductase. 

Verhagen, M.F.J.M., Kooter, I.M., Wolbert, R.B.G., and Hagen, W.R. 1993. On the iron-sulfur cluster of adenosine phosphosulfate reductase from Desulfovibrio vulgaris (Hildenborough). European Journal of Biochemistry 221 831-837. 

The first bacterial DPCK to be cloned and characterized was the E. coli CoaE protein coaE gene product, previously yacE), which was identified by a basic local alignment search tool (BLAST) using the N-terminal sequence of the wild-type enzyme purified from C. ammoniagenes The enzyme is a 22.6-kDa monomer in solution and exhibits apparent values of 140 pmol 1 and 740 pmol for ATP and dephospho-CoA, respectively. It displays poor activity with adenosine, AMP, and adenosine phosphosulfate (APS) as alternate phosphoryl acceptors, which all shows less than 8% activity compared with the natural substrate. Studies on the bifiinctional PPAT/ DPCK protein show that the normally reversible PPAT activity becomes irreversible when it is coupled to DPCK, most probably because the latter activity demonstrates a low K- for dephospho-CoA of 5.2 L5 pmol 1 (its for ATP was found to be 192 14 pmol 1 ). ° Similar values were determined in an independent study. ... 

Sulfate reduction reduction of the sulfote ion S04 " to the sulfide ion S, in which the hexavalent, positive sulfur of the sulfate is converted to the divalent, negative form. S.r. must be preceded by Sulfate activation (see). The substrate of enzymatic S.r. is therefore either adenosine phosphosulfate (APS) or phosphoadenosinephosphosulfate (PAPS). The enzy-mology of S.r. has been studied in particular in enzyme preparations from baker s yeast (Saccharomy-ces cerevisiae) and the anaerobic bacterium Desulfo-vibrio desulfuricans. The former organism performs assimilatory S.r. (see Sulfate assimilation), Ae latter dissimilatory S.r. (see Sulfate respiration). 

Peck HD, Deacon TE, Davidson JT. 1965. Studies on adenosine 5 -phosphosulfate reductase from Desulfovibrio desulfuricans and Thiobacillus thioparus. Biochim Biophys Acta 96 429 7. 

It has been proposed that 8-aminodcoxyguanosinc is formed from the nitronate tautomer of 2-nitropropane either by base nitrosation followed by reduction, or via an enzyme-mediated conversion of the nitronate anion to hydroxyiam ine-O-sulfonate or acetate, which yields the highly reactive nitrenium ion NHj (Sodum et al., 1993). Sodum et al. (1994) have provided evidence for the activation of 2-nitropropane to an aminating species by rat liver aryl sulfotransferase in vitro and in vivo. Pretreatment of rats with the aryl sulfotransferase inhibitors pentachlorophenol or 2,6-dichloro-4-nitrophenol significantly decreased the levels of nucleic acid modifications produced in the liver by 2-nitropropane treatment. Partially purified rat liver aryl sulfotransferase activated 2-nitropropane and its nitronate at neutral pH to a reactive species that aminated guanosine at the position. This activation was dependent on the presence of the enzyme, its specific cofactor adenosine 3 -phosphate 5 -phosphosulfate, and mercaptoethanol. It was inhibited... 

In Renilla the coelenterazine is stored as a coelenterazine sulfate, possibly having the structure shown. To convert this storage form to the active luciferin the sulfo group is transferred onto adenosine 3, 5 -bisphosphate to form 3 -phosphoadenosine 5 -phosphosulfate, the reverse of step d of Eq. 17-38. Tire luciferin of the ostracod crustacean Vargula hilgendorfii has a structure (Fig. 23-51) close to that from Renilla. 

Figure 2. Structure of the adenosine-5 -phosphosulfate (APS) molecule (Reproduced with permission from Ref. 9. Copyright 1986 U. Fischer).

Adenosine 5 -phosphosulfate was separated from ATP and other adenine nucleotides by chromatography on a Synchropak AX-100 column (4.1 mm x 250 mm). The mobile phase contained 0.1 M sodium phosphate buffer (pH 7.3) and 0.8 M NaHC03. The effluent profile was obtained by monitoring at 254 nm. 

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 9.141 Time course of formation of adenosine 5 -phosphosulfate (APS) by activity in rat liver. The reaction mixture contained, in a final volume of 350 /xL, 30 ju.mol of Tris-HCl (pH 8.0), 0.9 /xmol of ATP, 3 /unol of magnesium sulfate, 6 /xmol of sodium fluoride, and 50 fiL of inorganic pyrophosphatase (2.5 U). A50/xL supernatant sample (19 mg of protein) from rat liver was added to start the reaction. Then samples were removed at intervals, and the reaction was terminated and analyzed. Chromatograms were obtained after incubation for (A) 0 minutes, (B) 15 minutes, and (C) 45 minutes. Arrow indicates elution time for the reaction product APS. (From Mina and Rossomando, 1988.)...

Aryl sulfotransferases catalyze the transfer of the sulfuryl moiety from 3 -phosphoadenosine 5 -phosphosulfate to phenols, catechols, benzylic alcohols, arylhydroxylamines, and arylhydroxamic acids. This assay measures adenosine 3, 5 -diphosphate and is thus suited to quantitate enzyme activity when the sulfate esters formed are chemically unstable. 

Sulfate conjugation requires the prior activation of inorganic sulfate by adenosine triphosphate (ATP) to an active intermediate, 3 -phosphoadenosine-5-phosphosulfate (PAPSi. from which the sulfate group is transferred to a substrate (ROH). The final step is catalyzed by an enzyme called sulfotransferase. The three-step reaction sequence is illustrated in Figure 8.20. 

A.7.1 Esterification of Acids using Carbodiimides. The formation of anhydrides from carboxylic acids, thiocarboxylic acids, sulfonic acids and phosphorous acids are discussed in Section 2.4.S.2. In this section only special cases of anhydride formation are described. Mixed anhydrides of amino acids and adenylic acid are produced from the corresponding acids using DCC as the condensation agent. ° Mixed anhydrides not containing amino acids, such as butyryl adenate, adenosine 5 -phosphosulfate and p-nitrophenyl-thymidine-5-phosphate are also obtained. 

Figure 37-17 Schematic of pyrosequencing. individual dTNPs are added one by one to the single-stranded template, a primer, and a polymerase. Pyrophosphate is generated if the dNTP is complementary to the next base on the template (top). Any pyrophosphate produced reacts with adenosine-5 -phosphosulfate (APS) to produce ATP, which in turn generates light in the presence of Iuciferase (middle). The sequence can be determined from the order of dTNP addition and the intensity of light produced.

DNA polymerase I, Klenow Fragment, EXO(-) was obtained from Funakoshi (Tokyo, Japan). Luciferase, Adenosine-5 -triphosphate sulfurylase, adenosine 5 -phosphosulfate sodium salt, D-Luciferin sodium salt, and magnesium acetate Tetra hydrate were obtained from Sigma (MO, USA). Deoxynucleotide and 2 -deoxyadenosine 5 -0-(l-thiotriphosphate) were obtained from Amersham Pharmacia Biotech (UK). Other chemicals were of an analytical-reagent grade. Instruments ... 

Lyric RM, Suzuki I (1970c) Kinetic studies of sulfite cytochrome c oxidoreductase, thiosulfate-oxidizing enzyme, and adenosine-5 -phosphosulfate reductase from Thiobacillus thioparus. Can J Microbiol 48 594-603... 

Nucleoside 2 (or 3 ),5 -diphosphates have been isolated by degradation of certain coenzymes, as well as from hydrolyzates of nucleic acids. Adenosine 3, 5 -diphosphate (see p. 320) has been isolated by enzymic hydrolysis of coenzyme A and from active sulfate (adenosine 3 -phosphate 5 -phosphosulfate). Adenosine 2, 5 -diphosphate was shown to be present in the adenylic acid moiety of the coenzyme adenine-nicotinamide dinucleotide phosphate which, by treatment with a 5 -nucleotidase from potatoes, is converted into adenosine 2 -phosphate. Adenosine 3, 5 -di-phosphate is reported to play a role as a cofactor in the bioluminescence of Renilla reniformis (pansy) Ribonucleic acid carrying a terminal 5 -phos-phate group yields ribonucleoside 3, 5 -diphosphates on digestion with phosphoesterases. ... 

Stokes, A. M., Denner, W. H. B., and Dodgson, K. S., Kinetic properties of the soluble adenosine 5 -phosphosulfate sulfohydrolase from bovine liver. Biochim. Biophys. Acta 315, 402-411 (1973b). 

Fig. 31.41 The sulfoconjugation reaction example of 4-nitrophenol as acceptor substrate. Step 1, PAPS formation from adenosine 5 -phosphosulfate (APS) step 2, sulfate conjugation with the subsequent release of 3 -phosphoadenosine 5 -phosphate (PAP) step 3, possible hydrolysis of the sulfoconjugate by arylsulfatases.

A pathway first outlined in detail by Davies et al. (1966) has been examined in a number of tissues. Davies et al. (1966) used Euglena gracilis and showed that cysteic acid could act as a precursor of the sulfolipid. Incorporation from 801 was reduced in the presence of molybdate, which was thought to implicate adenosine-3 -phosphate 5 -phosphosulfate (PAPS). As a result of these experiments (see Table II), Davies et al. (1966) suggested a pathway involving formation of the sulfonic acid grouping by the reaction of PAPS with phosphoenolpyruvate. This reaction would be followed by a number of interconversions and, finally, by an aldol condensation with dihy-droxyacetone phosphate. 

M. are synthesized from the UDP-Af-acetylhexos-amine and UDP-n-glucuronic (or iduronic) add. Sulfate is transferred directly from adenosine-3 -phos-phate-5 -phosphosulfate after formation of the polysaccharide chain. Synthesis takes place in the endoplasmic reticulum. 

Renosto, F., Seubert, P. A., and Segel, 1. H. (1984). Adenosine 5 -phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization. J. Biol. Chem. 259, 2113-2123. 

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