Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.     

Inhibition of human placental sterylsulfatase by synthetic analogs of estrone sulfate

Synthetic analogs of estrone sulfate which carry differently substituted sulfonyl groups at position 3 of an invariable 3-desoxyestrone (dE1) moiety were tested in vitro as inhibitors of the human placental sterylsulfatase. Using both placental microsomes and a highly purified placental sterylsulfatase preparation as the enzyme source and dehydroepiandrosterone [³⁵S]sulfate or estrone [³⁵]sulfate as the substrate, the following order of inhibitory potencies was observed: dE1–3-sulfonylchloride >dE1–3-sulfonylfluoride≈dE1–3-sulfonate>dE1–3-sulfonamide≈3-methylsulfonyl-dE1. According to the results, the association of enzyme and inhibitor appears to be favored by an electronegative substituent at the sulfur atom (-Cl, -F, -O⁻). Since, however, even the most potent synthetic inhibitor was bound by the enzyme with significantly lower affinity than was the natural substrate estrone sulfate, an oxygen function between the aromatic ring and the sulfur atom may be necessary for high affinity binding towards the sterylsulfatase. In addition to its fast reversible association with the enzyme, dE1–3-sulfonylchloride further affected the sulfatase activity in a time-dependent manner. This latter inhibitory activity which may be due to a covalent modification (alkylation) of sterylsulfatase by the analog was partially prevented in the presence of substrate.     

Human placental sterylsulfatase: immunocytochemical and biochemical localization

Human placental sterylsulfatase was localised in situ by light and electron microscope immunocytochemical techniques as well as in homogenate and tissue extract fractions by enzyme assays. Light microscope observations on frozen sections of term and preterm placenta revealed sterylsulfatase immunoactivity primarily in the syncytiotrophoblast. Electron microscope observations confirmed the light microscope findings; in addition, they showed that the sulfatase is present in the endoplasmic reticulum of endothelial cells, too. In the syncytiotrophoblast, the enzyme was detectable in the cytoplasmic membrane of the nuclear evelope, in the membranes of the rough endoplasmic reticulum, in the plasma membrane with predominant localisation in coated pits, and in the membranes of endosomes and multivesicular bodies; little or no reactivity was detectable over the membranes of the Golgi complex and of lysosomes. Sterylsulfatase immunoactivity was absent in placentas with hereditary sterylsulfatase deficiency. The observations indicate that human placental sterylsulfatase is normally present in the membranes of compartments along the secretory pathway and the endocytic route of cells lining the fetal and maternal blood. Homogenates of normal term placenta as well as membrane vesicle preparations obtained by extraction of trophoblast tissue with isotonic saline were fractionated by differential centrifugation; the fractions were assayed for specific activities of sterylsulfatase and several marker enzymes of cellular topography. In agreement with our immunocytochemical findings, the results of these biochemical localisation experiments indicate the repeatedly described association of the placental sterylsulfatase with microsomal membranes but also point to the presence of the enzyme's activity in the microvillous plasma membrane of the syncytiotrophoblast. This localisation of sterylsulfatase may have functional implications in the placental uptake of circulating steroid sulfates.     

Origin of deoxycorticosterone and deoxycorticosterone sulfate in human pregnancy: absence of steroid 21-sulfatase activity in sulfatase-deficient placenta

The activity of steroid 21-sulfatase, the enzyme that catalyzes the hydrolysis of deoxycorticosterone sulfate (DOC-SO4) is demonstrable in human placenta. Thus, it is possible that this placental enzyme, by way of the hydrolysis of either DOC-SO4 or 21-hydroxypregnenolone mono- or di-sulfate of fetal origin, may be important in the biosynthesis of DOC, which is present in the plasma of pregnant women in high concentration. To investigate this issue further, we evaluated steroid 21-sulfatase activity in microsomal preparations of a sulfatase-deficient placenta. Immediately after delivery, at term, of a living male fetus with sulfatase deficiency, a microsome-enriched fraction of placental tissue was prepared; sulfatase activity was evaluated by use of three substrates, viz. dehydroisoandrosterone sulfate (DS), estrone sulfate (E1-SO4), and DOC-SO4, in various concentrations. Similar incubations were conducted with aliquots of a microsome-enriched fraction prepared from placental tissue of a normal fetus that was delivered, at term, within minutes of the time of delivery of the infant with sulfatase deficiency. In microsomal fractions from the normal placenta, each of the steroid sulfates was hydrolyzed. In the absence of microsomes, and in the presence of microsomal fractions from the sulfatase-deficient placenta, the hydrolysis of DOC-SO4 and DS was not detected. Moreover, in microsomes prepared from the sulfatase-deficient placenta, E1-SO4 was hydrolyzed at a rate that was only 10% of that in incubations with microsomal preparations of the normal placenta. We conclude that with sulfatase deficiency, the placenta is deficient not only in sulfatase activity for steroid-3-sulfates but for steroid 21-sulfates, e.g. DOC-SO4, as well.     

Preparative separation of alpha- and beta-naphthols catalyzed by immobilized sulfatase

It has been found that sulfatase from Helix pomatia hydrolyzes beta-naphthyl sulfate much faster than alpha-naphthyl sulfate; e.g., at pH 7.8, while the former is readily hydrolyzed, the latter undergoes no appreciable hydrolysis. Kinetic investigations of both enzymatic and acid hydrolysis of naphthyl sulfates and their analogs indicate that in the enzymatic reaction the difference in reactivities is due to steric hindrances exerted in alpha-naphthyl sulfate by the benzene ring adjacent to the one bearing the sulfate group. (In the beta-ester this ring is remote from the site of hydrolysis.) The enzyme was immobilized and employed for the preparative resolution of alpha- and beta-naphthols: a mixture of the isomers was first sulfated with chlorosulfonic acid and then incubated with sulfatase covalently attached to alumina. The beta-naphthol produced was extracted with benzene, followed by acid hydrolysis of alpha-naphthyl sulfate in the remaining aqueous solution and extraction of the alpha-naphthol formed. Helix pomatia sulfatase also expresses a marked regiospecificity in the hydrolysis of ortho and para substituted phenyl sulfates. Therefore, the enzyme can be used for the preparative separation of naphthols as well as a variety of isomeric phenols.     

The activity of arylsulfatase A and B on tyrosine O-sulfates.

L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.     

Properties of a bacterial enzyme preparation of alkylsulfatase

A preparation of primary alkyl sulfatase was obtained from the culture of Pseudomonas species 2T/1. It can hydrolyze alkyl sulfates, which belong to anion surface-active compounds, to sulfate ion and fatty alcohol, and as a result the harmful for biosphere property of the surface activity is gone. pH and temperature of the incubation mixture, the presence of ions of some bivalent metals and components of synthetic detergents (SD), composition of the buffer mixture and substrate concentration affect the rate of sodium dodecylsulfate (SDS) hydrolysis. The alkyl sulfatase preparation is relatively stable. The maximum rate of SDS hydrolysis was found to be at 70 degrees C. The preparation catalyzes the hydrolysis of some alkyl sulfate homologues and industrial alkyl sulfates. The temperature optimum of the preparation is 40 degrees C, the pH-optimum is 8.0-9.0.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

The N-terminal amino-acid sequence of human placental sterylsulfatase

The N-terminus of the recently isolated sterylsulfatase of human placental cellular membranes was sequenced. According to our results, the enzyme preparation proved to be homogeneous at least with respect to this part of the polypeptide chain; the n-terminal sequence of the sulfatase previously proposed by others, however, had to be revised partially.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

Lack of inhibition of placental estrone sulfatase and aromatase enzymes by vitamin D3 and its analogs

The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 125-dihydroxyvitamin D₃ and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D₃ and vitamin D₃ sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 μm. Only vitamin D₃ sulfate and 125-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 μm each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer cells in vivo.     

Primary Alcohol Sulfatase in a Pseudomonas Species

An ammonium sulfate-precipitated fraction from cell-free extracts of Pseudomonas C12B grown on a medium containing sodium dodecyl sulfate (SDS) contained alkyl sulfatase increased fourfold in specific activity over the crude. Optimal pH (7.5) and temperature (70 C) for sulfate release were determined with SDS labeled with radioactive sulfur (SDS(35)) as test substrate. Phosphate, arsenate, and certain heavy metal ions inhibited desulfation, whereas Mg(++) and Mn(++) stimulated activity of preparations which had been dialyzed against ethylenediaminetetraacetic acid. Dodecanol was recovered in semiquantitative yield from reaction mixtures containing enzyme and SDS(35). Aryl sulfates, secondary alcohol sulfates, and a phenoxyethyl sulfate failed to serve as substrate for this enzyme.     

Chemical characterization and substrate specificity of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) is a glycoprotein containing 4.6% carbohydrate in the form of 25 residues of mannose, seven residues of N-acetylglucosamine, and three residues of sialic acid per enzyme monomer of molecular weight 140 000. Each monomer consists of two equivalent polypeptide chains. The protein has a relatively high content of proline, glycine and leucine, and the amino acid composition of rabbit liver aryl sulfatase A is similar to that of other known liver sulfatases. Rabbit liver aryl sulfatase A catalyzes the hydrolysis of a wide variety of sulfate esters, although it appears possible that cerebroside sulfate is a physiological substrate for the enzyme because the Km is very low (0.06 mM). The turnover rate for hydrolysis of nitrocatechol sulfate or related synthetic substrates is much higher than the rate with most naturally occurring sulfate esters such as cereroside sulfate, steroid sulfates, L-tyrosine sulfate or glucose 6-sulfate. However, the turnover rate with ascorbate 2-sulfate is comparable to the rates measured using most synthetic substrates. These results are discussed in relationship to several previously described sulfatase enzymes which were claimed to have unique specificities.     

Cholesteryl sulfate and sterol sulfatase in the human reproductive tract.

Cholesteryl sulfate is a component of human seminal plasma (avg. 445 mug%) and spermatozoa (15 mug/10 (9) cells) and represents more than 85% of the sterol sulfate fraction. This conjugate is avidly bound by spermatozoa when compared to other steroids or steroid sulfates. Autoradiographic localization of CS associated with the spermatozoa revealed a greater accumulation of the radioactivity in the acrosomal region in many, but not all, of those cells examined. Semen is not a site of metabolism of the sterol sulfate but the enzyme, sterol sulfatase, is present in the human female reproductive tract. This cleavage enzyme was detected in Graafian follicles and the activity in the endometrium was ten-fold that found in the Fallopian tube. These findings lead to the proposal that cholesteryl sulfate, an amphipathic molecule ideally suited for interaction with membrane components and implicated in erythrocyte membrane stabilization, may be involved in membrane modifications of the spermatozoa during the process of fertilization.     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

Sulfate transport in human placenta: further evidence for a sodium-independent mechanism

Sulfate transport in isolated placental brush-border membrane vesicles has properties consistent with an anion exchange process. To ascertain the relevance of this finding to sulfate accumulation by the fetus and placenta in vivo, we examined sulfate transport in human placental tissue slices, comparing sulfate uptake with that of a non-metabolizable amino acid marker, alpha-aminoisobutyrate (AIB). In contrast to AIB, which was actively concentrated from physiological media, sulfate uptake by the placenta slice was concentrative only in the absence of sodium and at low pH. Uptake of sulfate reached a steady state after 60 min. It was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a specific inhibitor of anion transport, but not by ouabain. We found no evidence for Na(+)-dependent uptake of sulfate in incubated placental tissue. It seems unlikely that Na(+)-dependent sulfate transport by the placenta can be responsible for net sulfate accumulation by the human fetus.     

Purification and characterization of an inverting stereo- and enantioselective sec-alkylsulfatase from the gram-positive bacterium Rhodococcus ruber DSM 44541

Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (+/-)-2-octyl sulfate in a stereo- and enantiospecific fashion. When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30 degrees C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C(7) to C(10)) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates. Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X).