Purification and properties of the S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B

The S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B, was separated from other alkylsulphohydrolases and purified to homogeneity. Under the experimental conditions used the enzyme completely hydrolysed d-octan-2-yl sulphate (d-1-methylheptyl sulphate), but showed no activity towards the corresponding l-isomer. Additional evidence has been obtained to indicate that it is probably optically stereospecific for d-secondary alkyl sulphate esters with the ester sulphate group at C-2 and with a chain length of at least seven carbon atoms. Enzyme activity towards racemic samples of heptan-2-yl sulphate (1-methylhexyl sulphate), octan-2-yl sulphate and decan-2-yl sulphate (1-methylnonyl sulphate) increased with increasing chain length. l-Octan-2-yl sulphate is a competitive inhibitor of the enzyme, as are certain primary alkyl sulphates and primary alkanesulphonates. Inhibition by each of the last two types of compounds is characteristic of the behaviour of an homologous series. Inhibition increases with increasing chain length and plots of log K(i) values against the number of carbon atoms in each alkyl chain show the expected linear relationship. A crude preparation of the S2 secondary alkylsulphohydrolase was used to show that this particular enzyme hydrolyses l-octan-2-yl sulphate, but is probably inactive towards the corresponding d-isomer. The similarity of the S1 and S2 enzymes to the CS2 and CS1 enzymes respectively of Comamonas terrigena was established, and some comments have been made on the possible roles of these and other alkylsulphohydrolases in the biodegradation of detergents.     

Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B.

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.     

Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.     

Purification, properties and cellular localization of the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena.

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.     

Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Br?nsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.     

Hydrogen-bonding in enzyme catalysis. Fourier-transform infrared detection of ground-state electronic strain in acyl-chymotrypsins and analysis of the kinetic consequences.

I.r. difference spectra are presented for 3-(indol-3-yl)acryloyl-, cinnamoyl-, 3-(5-methylthien-2-yl)acryloyl-, dehydrocinnamoyl- and dihydrocinnamoyl-chymotrypsins at low pH, where the acyl-enzymes are catalytically inactive. At least two absorption bands are seen in each case in the ester carbonyl stretching region of the spectrum. Cinnamoyl-chymotrypsin substituted at the carbonyl carbon atom with 13C was prepared. A difference spectrum in which 13C-substituted acyl-enzyme was subtracted from [12C]acyl-enzyme shows two bands in the ester carbonyl region and thus confirms the assignment of the features to the single ester carbonyl group. The frequencies of the ester carbonyl bands are interpreted in terms of differential hydrogen-bonding. In each case a lower-frequency relatively narrow band is assigned to a productive potentially reactive binding mode in which the carbonyl oxygen atom is inserted in the oxyanion hole of the enzyme active centre. The higher-frequency band, which is broader, is assigned to a non-productive binding mode in each case, where a water molecule bridges from the carbonyl oxygen atom to His-57; this mode is equivalent to the crystallographically determined structure of 3-(indol-3-yl)acryloyl-chymotrypsin, i.e. the Henderson structure. A difference spectrum of dihydrocinnamoyl-chymotrypsin taken at higher pH shows resolution of a feature centred upon 1731 cm-1, which is assigned to a non-bonded conformer in which the carbonyl oxygen atom is not hydrogen-bonded. Perturbation of the protein spectrum in the presence of acyl groups is interpreted in terms of enhanced structural rigidity. It is reported that the ester carbonyl region of the difference spectrum of cinnamoyl-subtilisin is complicated by overlap of features that arise from protein perturbation. Measurements of carbonyl absorption frequencies in a number of solvents of the methyl esters of the acyl groups used to make acyl-enzymes have permitted determination of the apparent dielectric constants experienced by carbonyl groups in the enzyme active centre as well as a discussion of the effects of polarity. The ester carbonyl bond strengths of the various conformations were estimated by using simple harmonic oscillator theory and an empirical relation between the force constants and bond strengths. The fractional bond breaking induced by hydrogen-bonding was used to calculate rate enhancement factors by using absolute reaction rate theory.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of primary alkylsulphatases and metabolism of sodium hexan-1-yl sulphate by Pseudomonas C12B

Sodium hexan-1-yl sulphate and certain related alkyl sulphate esters have been shown to serve as inducers of the formation of primary alkylsulphatases (designated as P1 and P2) in Pseudomonas C12B. When the organism is grown on sodium hexan-1-yl [(35)S]sulphate as the sole source of sulphur or as the sole source of carbon and sulphur only the P2 alkylsulphatase is formed and inorganic (35)SO(4) (2-) is liberated into the media. Cell extracts contain this anion as the major (35)S-labelled metabolite although two unidentified labelled metabolites as well as choline O-[(35)S]sulphate occur in trace quantities in some extracts. Dialysed cell extracts are capable of liberating inorganic (35)SO(4) (2-) from sodium hexan-1-yl [(35)S]sulphate without the need to include cofactors known to be required for the bacterial degradation of n-alkanes. The collective results suggest that sodium hexan-1-yl sulphate can act as an inducer of P1 alkylsulphatase formation without the need for prior metabolic modification of the carbon moiety of the ester.     

The mechanism of action of primary alkylsulphohydrolase and arylsulphohydrolase from a detergent-degrading micro-organism.

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.     

Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties.

Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.     

Purification and some properties of the D-lactate-2-sulphatase of Pseudomonas syringae GG.

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.     

Lipids from the guinea pig Harderian gland: use of picolinyl and other pyridine-containing derivatives to investigate the structures of novel branched-chain fatty acids and glycerol ethers

The lipid extracted from guinea pig Harderian glands was hydrolysed and the constituents were examined as trimethylsilyl (TMS), (2H9)TMS, methyl ester/TMS, acetonide/TMS, nicotinate/TMS, picolinyl/TMS and nicotinylidene/TMS derivatives by capillary gas-liquid chromatography and gas chromatography/mass spectrometry. Over 70 compounds amounting to over 93% of the extract were identified. These consisted of 1-O-alkyl glycerols (glycerol ethers) with alkyl chains containing from 17 to 21 carbon atoms and fatty acids ranging from 14 to 26 carbon atoms. The alkyl chains in the glycerol ethers were straight, mono- and dimethyl-branched with the major site of branching being at C-14. All straight-chain acids from C14 to C26 were present, with the most abundant being n-24:0. Again mono- and dimethyl branched structures comprised the bulk of the remaining acids. Methyl groups tended to be towards the middle of the chain rather than in the more usual omega-1 (iso) and omega-2 (anteiso) positions, with C-14 again being a major site. The shorter-chain acids tended to have methyl groups closer to the acid group, with several of the short-chain compounds being substituted at C-2. Structural information on the acids was provided by the picolinyl derivatives and the sample provided an opportunity to evaluate these derivatives with branched acids other than the iso and anteiso compounds studied previously. They were found to be satisfactory for analysis of both mono- and dimethyl branched acids with the possible exception of compounds containing a methyl branch at C-4. However, in this case, structural information was provided by the methyl ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved procedure for the preparation of alkyl sulphate esters suitable for the study of secondary alkylsulphohydrolase enzymes.

A simple, rapid and convenient method for the synthesis of secondary alkyl sulphate esters is described in which the sodium alkoxide of the parent alcohol is sulphated by using triethylamine-SO3 complex. The procedure gives relatively good yields, even for the sulphation of long-chain alcohols and those in which the hydroxy group is remote from the terminal carbon atoms. Positional isomerization, arising from the migration of the hydroxy group along the carbon chain, is absent, and resolved enantiomers of alcohols react with complete retention of configuration.     

Inhibition of yeast pyruvate decarboxylase by alkyl phosphates]

It is found that yeast pyruvate decarboxylase is inhibited by alkyl phosphates. Inhibition is competitive with respect to a substrate. The inhibition constants with n-butyl and n-heptyl esters of phosphoric acid are the values of the same order of magnitude. With an increase in the length of the alkyl phosphates hydrocarbon chain from 7 to 10 carbon atoms inhibition constants change drastically. For n-heptyl phosphate and n-decyl phosphate values KI are equal to 1.6 x 10(-4) M and 1.7 x 10(-6) M, respectively. A further increase in the number of carbon atoms in the alkyl substituent of phosphoric acid ester induces no reduction of the inhibition constant. Multiple-inhibitor experiments of pyruvate decarboxylase show that inorganic phosphate and n-decyl ester of phosphoric acid are mutually exclusive. It is suggested that the inhibition mechanism with alkyl phosphates includes the competition of the phosphoric acid residue with alpha-ketocarboxyl group of pyruvate as well as the interaction between a hydrocarbon radical and hydrophobic parts on the enzyme surface, one of them being outside the substrate binding site.     

Highly diastereoselective addition of silyldihalomethyllithiums to chiral alkyl esters

Treatment of benzoate, formate, or trifluoroacetate esters with silyldibromomethyllithiums provides alkyl silyl mixed acetals via the 1,3-rearrangement of a silyl group from carbon to oxygen. A high level of asymmetric induction onto the acetal carbon is observed when chiral alkyl esters are employed. The stereochemical assignment of the silyl acetal 13j was accomplished on the basis of X-ray crystallographic analysis. A one-pot synthesis of a three-component coupling product R(1)C(OR(2))(OSiMe(2)-t-Bu)CX(2)E' (X = Cl, Br) by sequential additions of an ester (R(1)CO(2)R(2)) and the second electrophile was achieved.     

An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters.

Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.     

Glycosulphatase from Pseudomonas carrageenovora. Purification and some properties

A glycosulphatase present in the soluble fraction of disrupted Pseudomonas carrageenovora has been purified 500-fold by gel filtration on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose CL-6B. By dodecylsulphate/polyacrylamide gel electrophoresis the enzyme is practically homogeneous and has a molecular weight of 55 000. Conditions of optimal sodium chloride concentration and pH at 25 degrees C were 0.25--0.50 mol dm-3 and pH 7.0 respectively. The purified enzyme was inhibited by inorganic phosphate. Preparation is described of neocarrabiose 4-O-[35S]sulphate and neocarratetraose 4-O-[35S]sulphate from labelled Chondrus crispus. The purified glycosulphatase is active against both these substrates although only one of the two sulphate esters in the tetrasaccharide is hydrolysed. Analysis of the reaction products was by gel filtration, electrophoresis and 13C nuclear magnetic resonance spectroscopy. The results are consistent with the products of desulphation being respectively neocarrabiose and neocarratetraose 4-O-monosulphate with the sulphate ester proximal to the reducing end [3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-D-galactose 4-O-sulphate].     

Specificity of P2 primary alkylsulphohydrolase induction in the detergent-degrading bacterium Pseudomonas C12B. Effects of alkanesulphonates, alkyl sulphates and other related compounds.

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.     

Beef liver esterase. II. Kinetic properties

The kinetic parameters, k_cat and K_M, in beef liver esterase-catalyzed hydrolysis were determined for about 100 substrates, which can be classified in several groups: (1) In the ethyl ester series of fatty acids K_M decreases with elongation of the acid, while k_cat has a maximum value with pentanoate. (2) Alkyl acetates are better substrates as the alkyl moiety is longer, whereas esters with branched alkyl groups become worse substrates. (3) Aryl esters are very good substrates. (4) Esters of dicarboxylic acids are good substrates, but only one ester group is cleaved by the enzyme. Fumarate diester is susceptible to esterase hydrolysis, while maleate is not. (5) Esters of hydrophobic amino acids are very good substrates; the enzyme is not stereoselective and both the l and d stereoisomers are readily hydrolyzed. Branching at the β-carbon atom leads to loss of activity, and blocking of the amino group abolishes it. Fluoride ion and dl-malate esters are potent competitive inhibitors of the enzymic reaction. The optimal pH was found to lie between 8 and 8.5. The reaction rate increased between 5 and 40 °C then dropped sharply. The activity decreased at high salt concentration.     

Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket approximately 12 A deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.     

Synthesis of mono- and diglucosiduronates of metabolites of deoxycorticosterone and corticosterone and analysis by a new mass spectrometric technique

By condensing 3 alpha,21-dihydroxy-5 beta-pregnan-20-one, or its appropriate monoacetate, with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate in the Koenigs-Knorr reaction beta-D-glucosiduronates 10, 4, and 7 were obtained as polyacetate methyl esters. Alkaline hydrolysis of these substances cleaved the ester groups and gave the corresponding steroidal glucosiduronic acids 12, 6 and 8. Upon treatment with diazomethane, these acids produced the equivalent methyl esters. The C-3, the C-21 and the C-3,21 glucosiduronates of 3 alpha,21-dihydroxy-5 beta-pregnan-11,20-dione were prepared by previously reported methods and converted into the corresponding C-20 semicarbazones (14, 20 and 26). With C-20 stabilized by the semicarbazone group against reduction, it was possible to reduce the 11-oxo function in these substances to an 11 beta-hydroxyl group; after removal of the semi-carbazone moiety from these products at pH 2.0, glucosiduronic acids 18, 22 and 28 were obtained. The mass spectra of a representative group of the mono- and diglucosiduronic acids and esters were determined by utilizing fast atom bombardment and monitoring ions in both positive and negative modes of operation.