Bacteria isolated from soil, canal water, and sewage utilised short-chain (C1–C4) primary alkyl sulphate esters as sources of carbon and energy. Butyl and propyl sulphates, but not the C1 and C2 homologues, were degraded by a soil isolate (coryneform rod, strain B1a) which liberated SO2−4 stoicheiometrically from growth-supporting esters. Cell-extracts contained a sulphatase active towards C3–C7 primary alkyl sulphates, and this enzyme was presumed responsible for initiating the degradation. A sewage isolate (Xanthobacter sp. E5a) utilised exclusively ethyl sulphate, and a canal water isolate (strain M3C, tentatively assigned to the genus Agrobacterium) used only methyl sulphate. Strains E5a and M3c liberated SO2−4 stoicheiometrically from ethyl and methyl sulphates, respectively, but efforts to detect corresponding sulphatases in cell extracts failed, suggesting that metabolism of the C1 and C2 esters was initiated by an alternative, possibly oxidative, step. A Hyphomicrobium strain isolated from soil grew either aerobically or as a denitrifier in methyl sulphate minimal medium.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.

A highly purified commercial preparation of bovine testicular hyaluronidase (GL enzyme, Hyalosidase) was labelled with 125iodine without measurable loss of enzyme activity. The labelled preparation was administered intravenously into rats and the serum half-life of hyaluronidase was determined by measurement of both radioactivity and enzyme activity. The short half-life of the enzyme in plasma could not be accounted for by excretion in the urine and bile. Tissue distribution studies showed that the major site of uptake was the liver (59.7% of the recovered dpm). This rapid uptake by the liver could be reduced significantly by the pre-administration of yeast mannan o− ovalbumin (a mannose-terminated glycoprotein). This suggests that the uptake of hyaluronidase by the liver is mediated by a mannose-specific receptor. Very little radioactivity was found in the heart (0.2% of the recovered dpm).

A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.

Enrichment cultures from river sediments yielded several non-fermentative denitrifying bacteria, capable of anaerobic respiratory growth on the surfactant sodium dodecyl sulphate (SDS) as sole source of carbon and energy. Four selected isolates grew aerobically or anaerobically (with added nitrate) on primary alkyl sulphates of chain-length C6-C12 but not on the shorter homologues (C2-C5), or dl-octan-2-yl sulphate, or sodium dodecyltriethoxy sulphate. Cell extracts from aerobic or anaerobic cultures of the most prolific isolate contained a single major inducible alkylsulphatase active towards octyl sulphate but inactive towards propyl, butyl, and pentyl sulphates, and secondary alkyl sulphates.

The activation of the preemergent herbicide 2-(2,4-dichlorophenoxy)ethyl sulfate (Crag herbicide) is initiated by soil microorganisms that are presumed to act by removing the ester sulfate group via some type of sulfatase enzyme. An enrichment technique with the herbicide as the sole source of sulfur led to the isolation of several pure cultures that could produce 2-(2,4-dichlorophenoxy)ethanol from the herbicide. One of these, a strain of Pseudomonas putida , was particularly active. Polyacrylamide gel zymograms of extracts of cells grown on nutrient broth showed the presence of three secondary and three primary alkylsulfatases. One of the latter enzymes was active toward Crag herbicide as well as sodium dodecyl sulfate. Maximum activity was obtained in the late-stationary phase of growth, and enzyme yields were not affected by either the presence or the absence of the herbicide in the growth medium. The enzyme was purified 2,670-fold to homogeneity by a combination of streptomycin sulfate treatment, heat treatment, and column chromatography on DEAE-cellulose, Sephacryl 200-S, and butyl agarose. The pure enzyme was tetrameric (molecular weight, 295,000) and most active at pH 6.0. Saturation kinetics with inhibition by excess substrate were observed for Crag herbicide and octyl sulfate. 2-Butox-yethyl sulfate was a relatively poor substrate, and dodecyltriethoxy sulfate was not hydrolyzed at all. Enzymatic hydrolysis of each substrate in the presence of H 2 18 O led to incorporation of 18 O exclusively into SO 4 2− ions in all three cases. The Crag herbicide sulfatase therefore acts by cleaving the O-S bond of the C-O-S ester linkage, in contrast with other alkylsulfatases acting on long-chain alkyl sulfates.

1. A comparison was made of the metabolism of potassium D-(+)-octan 2-[35S]sulphate and potassium L-(-)-octan-2-[35S]sulphate in the rat. 2. Following administration of either enantiomer orally or i.v. the major proportion of the radioactivity was excreted in the urine within 24 h. When either enantiomer was administered i.v. to rats with bile-duct and ureter cannulae, the majority of the radioactivity was eliminated in the urine within six hours with only small amounts in bile. 3. Both enantiomers were extensively degraded in vivo. The metabolic products were identical with those previously reported (Maggs et al. 1982). 4. The major difference in the metabolite patterns was with respect to the relative amounts of hexanoate-5-sulphate: male and female urines contained approx. twice as much of this metabolite when the D-(+)-isomer was administered. In addition, isomer and sex-linked differences were observed with respect to the amounts of octanoate-7-sulphate.

1. The metabolism of potassium [2-14C]octan-2-sulphate and potassium octan-2-[35S]sulphate was investigated in the rat. Following oral administration, the bulk of the radioactivity was eliminated in the urine within 24 h. 2. Whole-body radioautography showed the liver to be the principal site of tissue accumulation of radiolabel following administration of 14C- or 35S-labelled DL-octan-2-sulphate. 3. Octan-2-sulphate was extensively degraded in vivo. The major urinary components are five sulphate estes, present in urine in essentially the same proportions regardless of label. The relative proportions of radioactivity associated with the urinary components showed considerable differences between male and female rats. 4. Three of the components have been identified as butanoate-3-sulphate, hexanoate-5-sulphate and octanoate-7-sulphate. The remaining metabolite was tentatively identified as an aldehyde derivative of octan-2-sulphate, a possible intermediate in the formation of octanoate-7-sulphate.

The P2 primary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200-250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a mol. wt. of 160 000. Activity towards primary alkyl sulphate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulphate substrates (C6-C12), logKm decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5kJ/mol for each additional CH2 group in the alkyl chain. logKi for the competitive inhibition by secondary alkyl 2-sulphate esters followed a similar pattern (-2.4kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. logKi values for inhibition primary alkanesulphonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accomodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulphate group, were not detectable at the active site.

Extracts of the cells of Comamonas terrigena, grown under a variety of different conditions, contain two distinct, constitutive, NAD-dependent alcohol dehydrogenase enzymes that can be separated by polyacrylamide-gel electrophoresis. One of the enzymes exhibits activity towards D-alkan-2-ols and primary alcohols and the other is active towards L-alkan-2-ols, symmetrical secondary alcohols and probably other positional isomers of secondary alcohols of the L-configuration. Methods for the individual assay of the two enzymes have been developed and have been used to define some of their general properties. Most of the substrates for these enzymes would not support growth of C. terrigena under the experimental conditions used and were relatively poorly oxidized by resting cell suspensions.

Primary alkanesulphonates were shown to serve as non-metabolizable (gratuitous) inducers of the P2 primary alkylsulphohydrolase enzyme in resting cell suspensions of Pseudomonas C12B. The effects of increasing concentrations of inducer on the production of enzyme were complex and suggestive of a multiphasic phenomenon. However, it was possible to determine Kinducer constants (analogous to Km or Ki) for alkanesulphonates of chain length from C7 to c12. these decreased with increasing chain length in a manner characteristic of an homologous series. Primary alkyl sulphates also served as good inducers of alkylsulphohydrolase, but valid kinetic values could not be obtained because these esters are good substrates for the enzyme and are therefore appreciably hydrolysed during the induction period. Small amounts of enzyme were also produced when cyprinol sulphate, dodecyltriethoxy sulphate C12H23-[O-CH2-CH2]3-O-SO3-Na+), Crag herbicide and some secondary alkyl sulphates were tested as inducers.

A series of d-alkan-2-yl sulphate esters (C(7)-C(14)) were prepared by sulphation of the resolved parent alcohols by a method that entails complete retention of configuration. These sulphate esters were tested as substrates for the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena. V(max.) reached a maximum with the C(9) compound, whereas logK(m) decreased linearly as the alkyl-chain length was increased from C(7) to C(14). A parallel series of l-alkan-2-yl sulphates was also prepared, and these esters, together with homologous series of primary alkyl sulphates and primary alkanesulphonates, were shown to be competitive inhibitors of the CS2 enzyme. For each series of compounds, logK(i) values decreased linearly with increasing alkyl-chain length. Plots of chain length against the standard free energy of binding (DeltaG(0)) of substrate and inhibitors to the CS2 enzyme showed that the standard free energy of association of a -CH(2)- group with the enzyme was 2.0-2.4kJ/mol for all classes of compound studied, indicating an important contribution from hydrophobic interactions to the overall binding. Plots for d-alkan-2-yl sulphate substrates and primary alkyl sulphate inhibitors were nearly coincident, suggesting that the overall interaction between a primary ester and the enzyme is the same as that between the isomeric secondary substrate and the enzyme. Plots for l-alkan-2-yl sulphate and alkanesulphonate inhibitors were very similar to each other, but were displaced by 1.5-3.0kJ/mol from that for substrate binding. This indicates that the binding of any one of these particular inhibitors involves one carbon atom fewer than the number involved in binding a substrate of the same chain length. These observations are discussed in terms of a three-point attachment of substrate to the enzyme involving the alkyl chain, sulphate group and the C-1 methyl group.

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000–46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C–O bond of the C–O–S ester linkage was confirmed by 18O-incorporation studies.

During the course of the purification of novel stereospecific secondary aklylsulphohydrolases present in certain detergent-degrading micro-organisms, it became apparent that substrates prepared by sulphating secondary alcohols with H2SO4 are heterogeneous. Apart from the racemization that occurs if resolved alcohols are sulphated, evidence is provided to show that other isomers are produced in which the position of the ester sulphate group on the alkyl chain has been altered. These changes can be avoided if pyridine/SO3 reagent (prepared with SO3) is substituted as sulphating agent. Experiments in which secondary alkyl sulphates prepared by both methods were tested as potential substrates for the two secondary alkylsulphohydrolase enzymes of Comamonas terrigena have provided initial information about the specificity of the enzymes.

The availability of homogeneous samples of the potassium salts of L- and D-octan-2-yl sulphate has enabled the separation of the optically stereospecific CS1 and CS2 secondary alkysulphohydrolases from extracts of cells of Comamonas terrigena. The CS2 enzyme was purified to homogeneity, and an initial study was made of its general properties, specificity, cellular localization and relationship to the CS1 enzyme. The CS2 enzyme has a molecular weight of approx. 250000 and a subunit size of approx. 58000, indicating that the molecule is a tetramer. Under the experimental conditions used the enzyme appears to be specific for (+)-secondary alkyl sulphate esters with the sulphate group at C-2 and with a chain length of at least six carbons. Enzyme activity towards racemic C-2 sulphates increases with increasing chain length up to C10, and there is some indirect evidence to suggest that activity declines when that chain length is exceeded. Other indirect evidence confirms that the CS1 enzyme exhibits similar specificity, except that only (-)-isomers can serve as substrates. Both enzymes are present in broth-grown stationary-phase cells of C. terrigena in approximately equal amounts.

The hydrolysis was studied of potassium (+)-octan-2-yl sulphate by two analogous, optically stereospecific, secondary alkylsulphohydrolases purified from two detergent-degrading micro-organisms, Comamonas terrigena and Pseudomonas C12B. Polarimetry studies have shown that (+)-octan-2-yl sulphate prepared from (+)-octan-2-ol is hydrolysed by both enzymes to yield (-)-octan-2-ol. This inversion of configuration implies that the enzymes are catalysing the scission of the C-O bond of the C-O-S linkage, a type of bond scission apparently not hitherto encountered among hydrolytic enzymes acting on ester bonds. Enzymic hydrolysis of potassium (+)-octan-2-yl sulphate in the presence of H218O and analysis of hydrolysis products for the presence of 18O has confirmed that C-O bond scission (and not O-S bond scission) occurs with both enzymes.

Previous studies have shown that secondary alkylsulphohydrolases from certain detergent-degrading micro-organisms are unusual esterases in that they catalyse fission of the C-O bond of the alkyl sulphate ester linkage. The position of bond fission catalysed by a primary alkylsulphatase and an arylsulphohydrolase present in Pseudomonas C12B has now been investigated. The primary alkylsulphatase behaved like the secondary alkylsulphohydrolases in cleaving the C-O bond of potassium heptan-1-yl sulphate. In contrast, the arylsulphohydrolase, in common with other similar enzymes previously studied, catalysed the fission of the O-S bond of potassium p-nitrophenyl sulphate.