The inhibition of fluoride of sulphatase A from ox liver and of the sulphatases of Helix pomatia and Aspergillus oryzae is decreased by EDTA and increased by Al3+ or Be2+, implicating aluminofluorides and beryllofluorides in the reaction. The inhibition, which is reversible, takes several minutes to develop fully and, at least for the sulphatase of H. pomatia, is of a non-linear mixed competitive-non-competitive type. It is suggested that the aluminofluorides and beryllofluorides are acting as analogues of HSO4-. If so, then this behaviour must be considered, as well as their role as analogues of phosphate, in interpreting the effects of these compounds in intact cells.

Correspondence| August 01 1989 The anomalous kinetics of sulphatase A A B Roy; A B Roy †Protein Chemistry Group, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia. Search for other works by this author on: This Site PubMed Google Scholar T J Mantle T J Mantle ‡Biochemistry Department, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1989) 261 (3): 689–697. https://doi.org/10.1042/bj2610689 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation A B Roy, T J Mantle; The anomalous kinetics of sulphatase A. Biochem J 1 August 1989; 261 (3): 689–697. doi: https://doi.org/10.1042/bj2610689 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1989 London: The Biochemical Society1989 Article PDF first page preview Close Modal You do not currently have access to this content.

1.1. The purification (57% total recovery) of two arylsulphatases from the digestive juice of the snail Helix pomatia is described.2.2. Both sulphatases are free from β-glucuronidase and N-acetyl-β-glucosaminidase, and electrophoresis on acrylamide gels detects no protein other than that associated with the arylsulphatase activity.3.3. Gel isoelectric focussing shows five protein bands (pH 3.9–4.8), all associated with arylsulphatase activity, in both fractions.4.4. The arylsulphatases have a wide specificity, with considerable variation in Km and V, and are inhibited by sulphate and sulphite.5.5. The variation of Km, Ki and V with pH is described: pKm varies linearly with pH between pH 5–9 (slope −1).6.6. The arylsulphatase and steroid sulphatase activities of the preparations are associated with a single protein.

The kinetic behaviour of the system native--substrate-modified sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) has been investigated and it has been shown that the progress curve of the complete reaction, including both the inactivation and reactivation stages, can be treated as that of a simple hysteretic system in which the substrate-modified enzyme is activated by a product of the reaction. It has been concluded that the early suggestions that the modification of sulphatase A was accompanied by the exposure of a second ligand-binding site are incorrect. It has been shown that, in the absence of sulphate, the rate of reversion of substrate-modified to native sulphatase A is increased by 4-nitrocatechol but not by the same concentration of 2-nitrophenol. A detailed reaction sequence is proposed. This explains the kinetic behaviour of sulphatase A with nitrocatechol sulphate or 2-nitrophenyl sulphate as substrate and in the presence or absence of sulphate.

The use of 17O-NMR to investigate bond cleavage during the hydrolysis of sulphate esters in water enriched in 17O is described. Despite the inherent disadvantages of 17O for NMR studies, this work shows that, in favourable cases, 17O-NMR of 17O-enriched species is a powerful and sensitive tool for mechanistic studies. It is particularly useful when OS cleavage occurs, resulting in the formation of S17O16O32− (5% 17O), which can easily be detected at the biologically relevant μmole level. The method complements those using H218O and has the advantage that in principle 17O can be detected in either of the hydrolysis products with little or no purification. It has been shown that sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) cleaves the OS bond while functioning as a cerebroside sulphatase, as it does when functioning as an aryl- or glycosulphatase.

The rhodizonic acid method for the determination of SO2-4 has been used to investigate the glycosulphatase activity of the sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver. Sulphatase A hydrolyses D-glucopyranose and D-galactopyranose 2-, 3-, 4- and 6-sulphates: glucose sulphates are hydrolysed more rapidly than galactose sulphates and the 3-sulphates more rapidly than the other isomers. 2-Acetamido-2-deoxyglucopyranose 6-sulphate is not hydrolysed, nor is 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose 1-sulphate. Sulphate is a competitive inhibitor of the glycosulphatase activity. Hydrolysis proceeds through fission of the O-S bond. Evidence is given that the hydrolysis of glucose 3-sulphate is accompanied by the formation of substrate-modified sulphatase A, although this has not been isolated. Sulphatase A has no detectable alkylsulphatase activity.

A simple apparatus for protonometry, the measurement of changes in proton concentration by changes in the potential of a glass electrode, is described. The simple theory of the method is considered, and its use is exemplified by the measurement of the initial velocities of a number of reactions catalyzed by sulfatases (EC 3.1.6.-).

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3′,5′-monophosphate (cyclic AMP) to adenosine 5′-phosphate at an optimum pH of approx. 4.3, close to that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP.On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. Sulphatase A also hydrolyses guanosine 3′,5′-monophosphate, but not uridine 3′,5′-monophosphate nor adenosine 2′,3′-monophosphate.

In a recent communication, A. Waheed and R. L. Van Etten (1979, Arch. Biochem. Biophys. 195, 248) showed that the sulfatase A of rabbit liver (arylsulfate sulfohydrolase, EC 3.1.6.1), which becomes inactivated as it catalyzes the hydrolysis of substrate, covalently incorporates 35S from nitrocatechol [35S]sulfate during this reaction and at the same time loses most of its secondary structure in solution. Circular dichroism spectra presented here for the native and turnover-modified forms of the sulfatase A of ox liver indicate no difference in the region of the spectrum below 240 nm associated with polypeptide backbone contributions or in the region from 350-250 nm associated with the side-chain chromophore transitions. In addition no differences were evident for the two forms of the ox liver enzyme from ultraviolet absorbance and fluorescence spectroscopy measurements. From these data we conclude that, in contrast to the situation with the rabbit enzyme, there is no loss of secondary structure associated with inactivation of ox liver sulfatase A in the course of enzymic catalysis.

Further studies have been made of the cerebroside sulphatase activity of the sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver. It is concluded that a cerebroside sulphate-modified form of the enzyme is not produced and that the kinetics of the reaction can be explained by the utilisation of the substrate and accumulation of (SO4)2-. The hypothesis is advanced that this difference between the cerebroside sulphatase and arylsulphatase activities arises from non-polar binding of the cerebroside to the enzyme. Possible reasons for the differences between these results and those of other (Stinshoff, K. and Jatzkewitz, H. (1975) Biochim. Biophys. Acta 377, 126-138) are considered.

The theoretical basis is given for methods of determining the apparent velocity constant, k∗, for the substrate-induced inactivation of sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) and the initial velocity, vo, of the catalytic reaction. The expression is of the same form as the empirical relationships previously used but the significance of the various terms is clearly established.

ATP sulphurylase (ATP:sulphate adenylyltransferase, EC 2.7.7.4) as been purified about 2500-fold from rat liver. It was free of ATPase, inorganic pyrophosphatase, adenosine phosphosulphate kinase and ADP sulphurylase activities. The enzyme was homogeneous to chromatography on Sepharose 4B and to density-gradient sedimentation; it was not homogeneous to acrylamide gel electrophoresis nor to sedimentation in the ultracentrifuge. Possible reasons for this heterogeneity are considered. The molecular weight of the enzyme is 410 000 as measured by chromatography on Sepharose 4B. The v is 0.80, suggesting that ATP sulphurylase is a lipoprotein. The enzyme activity is associated with a pigment having a lambdamax of 410 nm. Studies of the forward, reverse and ATP-PPi exchange reactions catalysed by ATP sulphurylase have shown that these are sequential bi-bi reactions, with ATP being the first substrate bound and adenosine phosphosulphate the last product released. The results are incompatible with previous suggestions that the ATP sulphurylase of rat liver catalysed a bi-bi ping-pong reaction.

The use of potassium 4-hydroxy-2-nitrophenyl sulphate (nitroquinol sulphate) as a substrate for the continuous spectrophotometric assay of arylsulphatase activity is described. It is hydrolysed by all arylsulphatases so far tested, including sulphatase B of ox liver. Optimum conditions for the assay of the sulphatases A and B of ox liver are described.

SummaryThe critical micelle concentration of cerebroside sulphate in water is 0·01 mM: it increases with increasing concentrations of buffer to 0·07 mM in 0·1 M sodium acetate and formate buffers. pH 5·6 and 4·5 respectively. The partial specific volume of the micelles is about 0·94. The behaviour of the micelles in the ultracentrifuge and on Sephadex G‐200 shows them to he grossly heterogeneous with respect to size. In 0·1 M buffer s 20, w is about 26 S; in water or 0·01 M buffer smaller micelles with an s20, w. of about 6 S are also present. In 0·01 M formate, pH 4·5, the smallest species detectable by equilibrium ultracentrifugation had a micellar weight of about 180,000 corresponding to an aggregation number of about 180. Much larger aggregates were also present.It is suggested that the smallest micelles are the substrate for sulphatase A when this is acting as a cerebroside sulphatase in buffers of low ionic strength.

Sulphatases B1alpha and B1beta (EC 3.1.6.1) have been prepared as apparently homogeneous proteins by chromatography on ConA-Sepharose. Both have a mol. wt. of 56 000, and E1%280nm of 17 and a turnover number of 8600 min-1 with nitrocatechol sulphate as substrate. Their amino acid compositions are identical: like sulphatase A, the sulphatases B are rich in proline and yield glucosamine on hydrolysis. They are not altered by treatment with neuraminidase. Both fractions show strong UDP-N-acetylgalactosamine 4-sulphatase activity, weak iduronate sulphatase activity, but no significant heparan N-sulphatase activity. It is suggested that the physiological activity of sulphatase B is that of the N-acetylgalactosamine 4-sulphatase which is lacking in the Maroteaux-Lamy Syndrome.

Weight-average elution volumes of sulphatase A (an arysulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 1028 to 1021 M−3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1 at any particular pH value studied it was relatively insensitive to temperature variation so that ΔH° is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A.

Ascorbic acid 2-sulphage has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates.