Arylsulfatases, substrates

The arylsulfatase substrate p-nitrophenyl sulfate is used together with lead nitrate in a manner analogous to the Gomori reaction (Example 1.5). 

Purified by dissolving in the minimum vol of hot water (60°) and adding EtOH, with stirring, then left at 0° for Ih. Crystals were filtd off and recrystd from H2O until free of Cl and SO ions. Dried in a vac over P2O5 at room temperature. It is a specific substrate for arylsulfatases which hydrolyse it to p-acetylphenol [A,max 327nm (e 21700 M- cm )] [Milsom et al. Biochem J 128 331 1972]. 

EC 3.1.6.1) is a lysosomal enzyme that hydrolyzes sulfuric acid ester bonds. The enzyme exists in two forms, arylsulfatases A and B, that differ in substrate specificity and in sensitivity toward inhibitors [142][143]. Human tissues contain more arylsulfatase A than arylsulfatase B. The natural substrates of these enzymes are complex lipids such as cerebroside 3-sulfate, and gly-cosaminoglycans such as chondroitin 4-sulfate and derman sulfate [144], Deficiencies of these enzymes are associated with a number of lysosomal disorders. 

A number of enzymes known as sulfuric ester hydrolases (EC 3.1.6) are able to hydrolyze sulfuric acid esters. They comprise arylsulfatase (sulfatase, EC 3.1.6.1), steryl-sulfatase (steroid sulfatase, steryl-sulfate sulfohydrolase, arylsulfatase C, EC 3.1.6.2), choline-sulfatase (choline-sulfate sulfohydrolase, EC 3.1.6.6), and monomethyl-sulfatase (EC 3.1.6.16). Whereas mono-methyl-sulfatase is highly specific and does not act on higher homologues, arylsulfatase has a broad substrate specificity and is of particular significance in the hydrolysis of sulfate conjugates of phenols, be they endogenous compounds, drugs, or their metabolites [167-169],... 

Studies on the fate of dopamine 4-sulfate (9.88) will serve to illustrate the reaction of deconjugation mentioned above. With this substrate, arylsulfatase activity in the dog was found to be highest in the kidney and liver, low in the intestine and heart, and almost nil in the brain and skeletal muscle [171]. Since this conjugate exists in high amounts in the plasma of humans, monkeys and dogs, the possibility was raised that it might be looked upon as a precursor or reservoir of free dopamine. 

Arylsulfatase [EC 3.1.6.1 ], also known simply as sulfatase, catalyzes the hydrolysis of a phenol sulfate, thereby producing a phenol and sulfate. This enzyme classification represents a collection of enzymes with rather similar specificities. (1) Steryl-sulfatase [EC3.1.6.2],also referred to as arylsulfatase C and steroid sulfatase, catalyzes the hydrolysis of 3-j8-hydroxyandrost-5-en-17-one 3-sulfate to 3-j8-hydroxyandrost-5-en-17-one and sulfate. The enzyme utilizes other steryl sulfates as substrates. (2) Cere-broside-sulfatase [EC 3.1.6.8], or arylsulfatase A, catalyzes the hydrolysis of a cerebroside 3-sulfate to yield a cerebroside and sulfate. The enzyme will also hydrolyze the galactose 3-sulfate bond present in a number of lipids. In addition, the enzyme will also hydrolyze ascorbate 2-sulfate and other phenol sulfates. 

For each patient sample, 10 and 20 pi of leukocyte homogenate are transferred to individual wells on the microtiter plate. As two different incubation times are necessary, the same volumes of all samples are also pipetted into the lower part of the plate. Subsequently, 100 pi of substrate buffer is added to each well and the volume is adjusted to a total of 150 pi by adding sodium acetate buffer containing barium acetate. Arylsulfatase A will be inhibited in the presence of barium ions. The plate is briefly shaken, sealed with tape, and covered with aluminum wrapping. Incubation is carried out at 37°C for 30 min (upper half including the standard curve) and 90 min (lower half). The reaction is stopped with 200 pi stop solution and the absorbance is read at 490 nm. 

All enzymes are measured at acidic pH with artificial fluorogenic substrates, except for arylsulfatase A, which is measured with a chromogenic substrate. The fluorogenic substrates are derivatives of 4-methylumbelliferone or 6-hexadecanoylamino-... 

Reiter et al. (43) have shown that a second enzyme can also act to degrade SGG. They found that secondary lysosomes from rat liver contained not only arylsulfatase A, but also a lipase activity that could act to de-acylate SGG. Under the conditions used, more product was formed by the action of the lipase on SGG than by the action of arylsulfatase A. These workers also found that the latter enzyme could use the lyso-SGG as a substrate. It would be... 

Arylsulfatase cleaves sulfate esters. Cerebroside sulfate is its natural substrate. The enzyme is also active toward p-nitrocatechol sulfate, which is the basis for this assay. 

The enzyme was prepared from human liver. This substrate is not hydrolyzed by arylsulfatase A. 

The HPLC method has been used to assay a number of activities usually found associated with lysosomal vesicles. All these assays utilize the fluorometric compound 4-methylumbelliferone (4-MU). When carbohydrates, lipids, phosphates, or sulfates are conjugated with 4-MU, these compounds can be used as substrates for glycosidase, lipases, acid phosphatases, and arylsulfatases. The activity is determined by the release of 4-MU. 

In the case of competitive inhibition the substrate and the inhibitor compete for the enzyme binding site. The same is true for product inhibition, where the accumulation of a product leads to a slow down of the enzyme reaction. Prominent examples are the inhibition of AP by phosphate, of arylsulfatase by sulfate, and of cholesterol oxidase by cholestenone. 

Mehl, E., and Jatzkewitz, H., Cerebroside 3-sulfate as a physiological substrate of arylsulfatase A. Biochim. Biophys. Acta 151, 619-627 (1968). 

Roy, A. B., L-Ascrobic acid 2-sulfate-A substrate for mammaiian arylsulfatases. Biochim. Bi-ophys. Acta 377, 356-363 (1975). 

Waheed, A., and Van Etten, R. L., Chemical characterization and substrate specificity of rabbit liver arylsulfatase A. Biochim. Biophys. Acta 614, 92-101 (1980a). 

It is also of interest to note that the GRE-activating enzymes are not the only radical SAM proteins that act on a protein substrate AtsB catalyzes the formation of a Cct-formylglycine (2-amino-3-oxopropionic acid, FGly) on AtsA, thereby activating this arylsulfatase (see Section 8.17.3.8). 

Since, as in simple Michaelis-Menten kinetics, the reaction rate depends linearly on the enzyme concentration, the accuracy of activator determinations depends on the precision with which the enzyme concentration is known. Although it is not necessary to employ pure enzymes for the purpose of activator quantification, problems may arise if several isoenzymes exist of which only one is able to degrade the glycolipid substrate in the presence of the activator but all of them hydrolyze the artificial substrates employed for measuring the enzyme. This is, for example, the case for a-galactosidase, p-hexosaminidase, and arylsulfatase. In these cases separation of the isoenzymes, say by ion-exchange chromatography, is required. 

Arylsulfatase activity is usually measured with the chromogenic substrate 4-nitrocatecholsulfate. (A fluorogenic substrate, 4-methylumbelli-ferylsulfate, is also available but is less frequently used because it is hydrolyzed only very slowly.) The assay system of Baum et al. (1959), which was developed to permit the determination of arylsulfatase A in the presence of the B isoenzyme, is still most widely used. The unusual time course of the hydrolysis of 4-nitrocatecholsulfate by arylsulfatase A (Roy, 1953 Baum et al., 1958 Stinshoff, 1972) may present a problem for standardization of the enzyme preparation and should be taken into account, such as by using short incubation times (15-30 min). The enzyme unit is usually defined as the amount of enzyme that hydrolyzes 1 p.mol of this substrate per minute. 

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.

Arylsulfatase ARS2 Chlamydomonas reinhardtii Davies et al. [29] and de Hostos et al. [55] Excreted into media and cleaves chromogenic substrates... 

Sulphur was found through spark source mass spectrometry to be abundant in SRS-A [93], Also, incorporation of S into SRS has been reported [64,94-96], The observation that several thiols enhance SRS formation in different systems [97-100] and that arylsulfatase (an enzyme that cleaves sulfate monoesters of phenolic and other unsaturated hydroxylated systems) inactivates SRS-A [101,102] opened the view that a thiol is a constituent of the active compound. However, it has recently been shown that the SRS-inactivating action of commercially available arylsulfatase is not due to sulfate ester cleavage, but to a dipeptidase contaminant [103,104]. The destruction of different SRS compounds by hpoxygenase [67,105,106] shows that SRS contains a cij,c/s-1,4-pentadiene structure, since this is a prerequisite for a lipoxygenase substrate [107], For a review of the earlier structural work on SRS, see ref. 108. 

There are no characteristic abnormalities of serum or of formed blood elements in ML. Non-specific hypercholesterolemia has been seen (Hagberg 1963), but sulfatides are present in normal amounts (Svennerholm and Svennerholm 1962). The ratio of cerebrosides sulfatides, which is abnormal in the central nervous system, is normal in serum (Hagberg 1963). Some relevant serum enzymes have been studied by Austin et al. (1965 a, b). According to these authors acid phosphatase activity was normal when measured with p-nitrophenylphosphate as substrate. The activity of arylsulfatase B was three to four times that of type A, and thus similar to findings in normal serum. 

In 1963, Mehl and Jatzkewitz succeeded in isolating fractions with cerebroside-sulfatase activity from pig kidneys, and Bleszynski and Dzialoszynski (1965) purified soluble arylsulfatases from ox brain. A study by Mehl and Jatzkewitz (1965) on the significance of a lack of arylsulfatase in ML as reported by Austin (1963 a), and Austin et al. (1964 a, b), with 2-hydroxy-5-nitrophenyl-sulfate as substrate, resulted in the finding of two fractions with arylsulfatase activity in normal kidneys. While in ML the smaller of both fractions was present in normal concentration, the second component, and the predominant one in normals, was below the limits (0.005 o. d. units Mehl and Jatzkewitz 1965) of the method in ML. It seems that diminution or lack of this heat-labile fraction, which corresponds to the arylsulfatase A of Austin, is typical for ML, and according to Mehl and Jatzkewitz (1965), supports the assumption of a block in the degradation of ML between sulfatides and cerebrosides (see fig. 3). 

Arylsulfates are decomposed by hydrolytic enzymes, a reaction similar to the hydrolysis of phosphoric acid esters by phosphatases (66, 66). It has been suggested that the true substrates of arylsulfatases are sulfuric acid esters of sterol hormones (67). 

The first activator protein was discovered in 1964 as a protein required for the hydrolytic degradation of sulfatides by lysosomal arylsulfatase A [18]. This sulfatide-activator or SAP-B (saposin B), binds GSLs, but with broader specificity than the GM2 activator. In vitro it behaves similarly to the GM2 activator in some aspects, i.e. it can present GSLs to water-soluble enzymes as substrates [19). 

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