Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides

BB

Biochimica et BiophysicaActa 1251 (1995) 11-16

ELSEVIER

Biochi~ic~a et BiophysicaAEta

Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides Srdanka Tomi6 a,*, Anda Tre~ec b, Jelka Toma~i6 b, Biljana Petrovi6 b,1 Vera Simeon Rudolf c, Mira Skrinjari6-Spoljar c, Elsa Reiner c v

v

a Department of Chemistry, Faculty of Science, University ofZagreb, Strossmayerov trg 14, 41000 Zagreb, Croatia b Institute of Immunology, Rockefellerova 10, 41000 Zagreb, Croatia c Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 41001 Zagreb, Croatia

Received 15 February 1995; accepted 28 March 1995

Abstract

Rabbit serum and one enzyme fraction isolated from rabbit serum by column chromatography (Fraction II) were used as catalysts in regioselective hydrolysis of r~Ldiolabelled pivaloylated monosaccharides (Piv = Me3CCO). The hydrolysis of ~4C-labelled methyl 2-O-pivaloyl-(2-MP)-, 6-O-pivaloyl (6-MP)-, 2,6-di-O-pivaloyl-(2,6-DP) a-D-glucopyranosides and methyl 2-acetamido-2-deoxy-3,6-diO-pivaloyl-(3,6-DPNAc) a-o-glucopyranosides, was studied, as well as that of the non-sugar substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. The specific activities of 2,6-DP, 3,6-DPNAc, butyrylthiocholine and thiophenylbutyrate were higher in Fraction II than in native sera, while those of phenylacetate and paraoxon were lower. Inhibition studies were done using the substrates mentioned and five different inhibitors, namely bis(p-nitrophenyl phosphate) (BNPP), eserine, paraoxon, HgCI 2 and EDTA. The hydrolysis of 2,6-DP and 3,6-DPNAc was not inhibited by HgC12 and only slightly by EDTA. Paraoxon, eserine and BNPP were progressive inhibitors of the hydrolysis of the two sugar substrates, and the pattern of inhibition resembled closely the inhibition of butyrylthiocholine and thiophenylbutyrate hydrolysis. This result applied to both, native serum and Fraction II. It was concluded that esterases in rabbit serum which hydrolyze pivaloylated sugar substrates belong to the category of serine esterases. Kinetic parameters (K M and Vmax), effects of temperature and pH on activity of esterases from Fraction II were also determined for the hydrolysis of sugar substrates. Keywords: Serum esterase; Esterase characterization; Pivaloylated sugar substrate; (Rabbit)

1. Introduction

During the last few decades the use of enzymes as catalysts in organic synthesis has developed into a very interesting field of investigation [1,2]. In addition to their stereoselective properties enzymes also offer the opportunity to carry out chemo-[3,4] and regio-selective transformations [5,6]. This is of spe,cial interest in dealing with multifunctional compounds in which one particular functionality has to be transformed in the presence of other functionalities of similar reactivity. Mono-and oligosaccharides, glycosides and other glycoconjugates belong to this class of multifunctional compounds since they embody a

* Corresponding author. Fax: +3~5 1 432526. I Present address: Organic Chemical Industry Skopje, 91000 Skopje (Macedonia). 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 ! 67-4838(95)0005 6-9

multitude of hydroxyl groups of comparable chemical reactivity. Consequently, for the directed construction of carbohydrate derivatives enzymatic techniques offer enormous advantages, and this field has been investigated intensively in recent years [5,6]. We have shown that acylated monosaccharides may be regioselectively transformed by enzymes present in mammalian sera [7,8]. The selective hydrolysis of various acylated sugars (cf. Fig. 1) was investigated using native mammalian sera or the esterases isolated from sera [9-14]. Although the commercially available enzymes, mostly lipases and some esterases were used in transformations of sugars, the catalytic capabilities of mammalian sera esterases specific for sugar substrates were not, to our knowledge, previously reported. Mammalian sera, which are a very rich source of the enzyme activities contain several groups of esterases which

12

S. Tomi{ et al. / Biochimica et Biophysica Acta 1251 (1995) 11-16 CH2R2

Ho~O~ RI ~'~\ ~ ~ ' ~ ' ~ \~R

pi, = (CH3)sCC0 l

Ac=CH3CO OMe

2-MP

R = OPiv

RI =

R 2 = OPiv

R 2 = OH

6-MP

R = R 1 = OH

2,6-DP

R = R 2 = OPiv

R I = OH

3-MPNAc

R =NAc,

R 2 = OH

3,6-DPNAc

R = NAc

R 1 = OPiv

R I = R 2 = OPiv

Fig. l. Structures of carbohydrate substrates and their respective hydrolysis products.

might be involved in the hydrolysis of acylated sugars. One of these groups are serine esterases, such as carboxylesterase (EC 3.1.1.1) and butyrylcholinesterase (EC 3.1.1.8) which preferentially hydrolyse aliphatic carboxylesters and are progressively inhibited by organophosphorus compounds and carbamates [ 15,16]. The other group of enzymes are arylesterases (EC 3.1.1.2) which primarily hydrolyse aromatic esters, and phosphoric triester hydrolases (EC 3.1.8.1) which hydrolyse fully substituted esters of phosphoric, phosphonic and phosphinic acids, and are inhibited by some heavy metal cations and chelating agents [15-21]. Each of these groups of enzymes are characterized by the hydrolysis of specific substrates, but all of them are capable to hydrolyse at lower rates also a variety of other esters. In this paper we report on some catalytic properties of the enzyme(s) hydrolysing acylated sugar substrates and evaluate their resemblance to the above stated esterases. The study was done on rabbit serum esterases which are known to have broad substrate specificities [22-24]. In addition to the acylated monosaccharides we also used as substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. In order to differentiate the esterases we have used the following inhibitors: eserine and paraoxon as inhibitors of serine esterases, BNPP as inhibitor of carboxylesterase and EDTA and HgCI 2 as inhibitors of paraoxonase. The same inhibitors were applied in the hydrolysis of acylated sugar substrates.

2. Materials and Methods 2.1. Materials Substrates. Methyl 2,6-di-O-pivaloyl-a-D-[U-14C]glucopyranoside (2,6-DP), methyl 2-acetamido-2-deoxy-3,6-diO-pivaloyl-a-D-[U-~4C]glucopyranoside (3,6-DPNAc), methyl 2-O-pivaloyl-a-D-[U-14C]-glucopyranoside (2-MP) and methyl 6-O-pivaloyl-a-D-[U-lac]glucopyranoside (6MP) were synthesized as described previously [9,11]: stock

solutions (31 mM) of 2,6-DP, 2-MP and 6-MP were prepared in dimethylsulfoxide (DMSO); stock solution (20 mM) of 3,6-DPNAc was prepared in ethanol (96%). Butyrylthiocholine iodide (BTC, Sigma); stock solutions (50 mM) and all further dilutions were prepared in water. Thiophenylbutyrate (TPB, Polysciences): stock solutions (150 mM) were prepared in methanol and further dilutions in phosphate buffer (0.1 M, pH 7.4). Phenylacetate (PA, Fluka): stock solutions (200 mM) were prepared in 40% ( v / v ) ethanol in water and further dilutions in water. Diethyl-4-nitrophenyl phosphate (Paraoxon, POX, Bayer): stock solutions (6.88 mM) were prepared in methanol and further dilutions in Tris-HCl buffer (0.1 M, pH 7.4). Inhibitors. l'-methylpyrrolidino(2':3':2:3)l,3-dimethylindoline-5-yl-N-methylcarbamate (eserine, Fluka), bis(pnitrophenylphosphate) (BNPP, Sigma), ethylene diamine tetraacetic acid disodium salt (EDTA, Serva) and HgC12 (Kemika): stock solutions (10 mM) and all further dilutions were prepared in water. Stock solutions of paraoxon (2.7 mM) in DMSO were prepared for assays with 2,6-DP and 3,6-DPNAc as substrates. Stock solutions of paraoxon (50 mM) were prepared in methanol, and further dilutions in Tris-HC1 buffer, for assays with BTC, TPB, and PA as substrates. Stock solutions of 2,6-DP, 3,6-DPNAc, 2-MP and 6-MP were kept at - 20°C. All other stock solutions were kept at 4°C. All dilutions were made before the beginning of the experiment. 2.2. Methods General Thin-layer chromatography (TLC) was performed on Silica gel 60 (Merck) using Solvent A: ethyl acetate/benzene/ethanol (5:1:1). Detection was effected by charring with sulfuric acid. Radioactivity was measured by using a Beckman LS-100C liquid scintillation counter and Aquasol (NEN) as a scintillation cocktail. Protein concentrations were determined by the method of Lowry [25], and by measuring the absorbance at 280 nm and using bovine serum albumin as standard. Spectrophotometrical measurements were done using Unicam SP500 and Cary/3 spectrophotometers; optical path was 1.0 cm. Rabbit serum. Blood was removed from rabbits (New Zealand white) by heart puncture, transferred to centrifuge tubes, stored for a few hours at room temperature, and then centrifuged at 3000 r.p.m, for 10 min. The supernatant serum was removed by aspiration and stored at -20°C. Preparation of partially purified enzyme. All procedures were carried out at 4°C. Unless stated otherwise 0.067 M phosphate buffer (pH 6.3) was used for equilibration of the gels and elution. After each step, the fractions that contained esterase activity were collected and concentrated by ultrafiltration using an Amicon Diaflo UM-10 membrane. Esterase activities were detected as described below.

S. Tomid et al. / B iochimica et Biophysica Acta 1251 (1995) 11-16

Rabbit serum (60 ml) was dialysed twice against phosphate buffer (2 1) and centrifuged at 100000 g for 1 h in order to remove the precipitate that did not contain esterase activity. The supernatant serum (62 ml) was applied to a column of DEAE-Sepharose CL-6B and chromatographed as previously reported [12]. The fractions were first eluted with phosphate buffer (280 fractions, 5 ml each) and then with 0.1 M NaC1 phosphate buffer (160 fractions, 5 ml each), followed by a linear gradient of NaC1 (0.1-1.5 M) in the same buffer. The enzyme activity was determined as described below in procedure c) using radiolabelled 2,6-DP as a substrate. Fractions 125-175 and 350-400 showing esterase activities were collected separately and concentrated by ultrafiltration. Two peaks were thus isolated, Fraction I as a result of concentration of fractions 125-175 and Fraction II as a result of concentration of fractions 350-400. Assay of enzyme activities (a) Hydrolysis of radiolabelled methyl 2,6-di-Opivaloyl-a-D-glucopyranosicle (2,6-DP) [9,12]. The assay medium contained phosphate buffered saline (PBS, 75 /zl, 0.01 M, pH 7.2), rabbit serum or enzyme Fraction II (50 /zl) and 2,6-DP (10 /zl in DMSO) as a substrate. The disappearance of 2,6-DP was monitored by TLC (Solvent A). The assay medium in inhibition studies contained PBS (65 /zl), rabbit serum or enzyme Fraction II (50 /zl), the respective inhibitor (10/xl) and 2,6-DP (10/zl in DMSO). (b) Hydrolysis of radiolabelled methyl 2-acetamido-2deoxy-3,6-di-O-pivaloyl-a-D-glucopyranoside (3,6DPNAc) [10,11]. The assay medium contained PBS (80 /xl), rabbit serum or enzyme Fraction II (50 /xl) and 3,6-DPNAc (6/xl in DMSO) as a substrate. The disappearance of 3,6-DPNAc was monitored by TLC (Solvent A). The assay medium in inhibition studies contained PBS (70 /zl), rabbit serum or concentrated enzyme Fraction II (50 /zl), the respective inhibitor (10/zl) and 3,6-DPNAc (6 /zl in DMSO). (c) A screening assay of enzyme activity during the purification procedure was performed on diluted fractions. The assay medium contained individual fractions (100-300 /zl depending on the expected activity), PBS (200-400 /zl), and J4C-labelled 2,6-DP (10 /zl in DMSO). Each resulting mixture had a volume of 500 /xl. Incubation times in (a) and (b) were 20 min unless otherwise stated. Incubation time for diluted fractions in screening assays (c) was 12 h. All assays were done at 37°C, the reaction mixtures were 1.0 mM with respect to the substrate, and the reactions were stopped by the addition of ethanol. The work-up procedure was followed as described previously [12]. ]inhibitions were measured at inhibition time zero and 30 min. The enzyme activity at zero-time inhibition was determined by adding the enzyme (rabbit serum or Fraction II) to the assay medium which contained all other reactants. Reactions were stopped 5

13

min after the addition of the enzyme. The enzyme activity at 30 min inhibition time was determined by adding the substrate to the assay medium after enzyme and inhibitor were preincubated for 30 min. Control samples contained PBS instead of the inhibitor solution. (d) Hydrolysis of butyrythiocholine (BTC) and thiophenylbutyrate (TPB) [26] (A = 412 nm; e M for 5-thio-2nitrobenzoic acid = 13 600 M - l c m - l ) . The assay medium contained phosphate buffer (2.0 ml), the thiol reagent DTNB (100 /xl, final concentration 0.33 mM), rabbit serum (final conc. 0.7 and 0.2% v / v , respectively) or Fraction II (300/zl), water or respective inhibitor (300/zl) and BTC or TPB (300 /zl). (e) Hydrolysis of phenylacetate (PA) [21,27] (A = 270 nm; e M for phenol = 1510 M - 1 cm- 1). The assay medium contained Tris-HC1 buffer (2.0 ml), rabbit serum (final conc. 0.7% v / v ) or Fraction II (300 ~1), water or respective inhibitor (300 /zl) and PA (300 /1,1). (f) Hydrolysis of paraoxon (POX) [21] (A = 405 nm; e M for 4-nitrophenol= 16000 M -j cm-~). The assay medium contained Tris-HC1 buffer (100 /zl), rabbit serum (final conc. 9.1% v / v ) or Fraction II (100 /xl), water or respective inhibitor (100 /xl) and POX (800 /zl). All activities in (d), (e) and (f) were measured spectrophotometrically at 37°C at pH 7.4. Enzyme inhibition in these experiments was measured at inhibition times zero and 30 rain. The enzyme activity at zero-time inhibition was measured by adding the enzyme (rabbit serum or Fraction II) to the assay medium which contained all other reactants; the increase in absorbance was read immediately thereafter. The enzyme activity at 30 min inhibition time was measured by adding the substrate to the assay medium after enzyme and inhibitor were preincubated for 30 min. Control samples contained water instead of the inhibitor solution. The increase in absorbance was read up to 2 min and it was linear with time in control and inhibited samples. and Vmax determinations Kinetic data were obtained for three radiolabelled carbohydrate substrates, namely 2,6-DP, 2-MP and 6-MP. Incubation mixtures contained Fraction II (50 /xl). PBS (75 /zl) and the relevant substrate in DMSO (10 /zl). The resulting mixtures were 0.50, 0.75, 1.0, 2.0, 4.0 and 5.0 mM with respect to the substrate. Reaction times varied according to the respective substrate concentrations. The disappearance of the respective substrate was monitored by TLC (Solvent A). Velocities of ester hydrolysis were determined as a function of substrate concentration. Kinetic calculations were done on an Apple Macintosh Ilci computer and values for K M and Vmax obtained by the Michaelis-Menten equation fitting. KM

2.3. Effect of pH on Fraction H activity The activity of Fraction II as a function of pH was measured with radiolabelled 2,6-DP as substrate at pH

14

S. Tomid et aL / Biochimica et Biophysica Acta 1251 (1995) 11-16

Table 1 Specific activities (mean+S.D.) of rabbit serum esterases in sera and Fraction 1I. Specific activities were determined in four pools of sera and in Fraction II obtained in four separation runs of the respective pools. The substrate concentrations are given in brackets

All incubation times were 10 min. The disappearance o f 2,6-DP was m o n i t o r e d by T L C ( S o l v e n t A).

Substrate

3. Results

Specific activity (nmol min- i mg- i ) Native sera (NS)

Fraction II (F II)

Ratio (F II/NS)

2,6-DP (1.0 mM) a 5 __+2 39 + 19 3,6-DPNAc (1.0 raM) b 4+0.5 34+7 Butyrylthiocholine (5.0 mM) 24 + 7 86 + 36 Thiophenylbutyrate (1.0 mM) 68 + 7 109 + 43 Phenylacetate (5.0 mM) 5900 + 2800 3700 + 2300 Paraoxon (5.0 mM) 31 + 32 10_ 6

7.8 8.5 3.6 1.6 0.63 0.32

a Methyl 2,6-di-O-pivaloyl-ot-D-[U-14C)gluocopyranoside. b Methyl 2acetamido-2-deoxy-3,6-di-O-pivaloyl-ot-D-[U- 14C]glucopyranoside.

values b e t w e e n 5.5 and 12.5. The final reaction mixture was incubated at 37°C for 10 min, and consisted o f P B S (75 /xL), Fraction II (50 /xL) and a solution of 2,6-DP in D M S O (10 ~ L ) . The resulting mixture was 1.0 m M with respect to 2,6-DP. The disappearance o f 2,6-DP was monitored by T L C ( S o l v e n t A). Parallel assays containing PBS instead o f Fraction II were p e r f o r m e d as controls. 2.4. E f f e c t o f t e m p e r a t u r e on F r a c t i o n H a c t i v i t y

The effect o f temperature on activity o f Fraction II was studied b e t w e e n 2 0 - 7 0 ° C with radiolabelled 2,6-DP as substrate, and at p H 7.2. A s s a y mixtures, prepared as a b o v e but without the substrate solution, were preincubated for 5 min at relevant temperatures. The reactions were initiated by the addition of the substrate in D M S O .

Rabbit serum is a rich source o f esterases including the esterase(s) with specificity for acylated sugars [9-14]. It was found, e.g., that the treatment o f laC-labelled 2,6-DP with native rabbit serum results in the r e m o v a l o f the 6-Piv group at m u c h faster rates than the 2-Piv group, and a 6:1 mixture of 2 - M P and 6 - M P was o b s e r v e d [19,12]. E v e n m o r e p r o n o u n c e d regioselectivity was o b s e r v e d in the rabbit serum catalyzed hydrolysis o f 3 , 6 - D P N A c where the m o n o p i v a l a t e 3 - M P N A c f o r m e d almost exclusively [ 11,12]. C o l u m n c h r o m a t o g r a p h y o f rabbit serum on D E A E Sepharose C L - 6 B g a v e two peaks that contained esterase activity based on the use of 14C-labelled 2,6-DP as a substrate [12]. The first peak contained approx. 5% of the total activity applied. The rest of the activity associated with this esterase (Fraction I) o v e r l a p p e d with serum albumin and was disregarded in further experiments. E n z y m e Fraction I caused n o n - s e l e c t i v e hydrolysis of both pivaloyl groups f r o m 2 , 6 - D P and g a v e a 1:1 mixture of m o n o p i valates 2 - M P and 6-MP. Esterase Fraction II was eluted with 0.1 M NaCI in buffer and contained approx. 75% of the activity applied. Fraction II catalyzed preferentially the hydrolysis of the 6-Piv group to give 2-MP. O n l y after a considerable prolongation o f reaction times (up to 48 h) a small proportion of the 6 - M P was observed. The same result was obtained w h e n 3 , 6 - D P N A C was used as a substrate, and 3 - M P N A c f o r m e d as a sole product of hydrolysis catalyzed by Fraction II. E n z y m e Fraction II,

Table 2 Inhibition (%) of rabbit serum esterases. POX (10 nM), BNPP (10 /xM), eserine (10 /xM), EDTA (1.0 mM) and HgCI 2 (10/xM) were used as inhibitors. The substrate concentrations were the same as in Table 1 Substrate

Inhibitor POX BNPP Time of inhibition (min) zero

Native serum 2,6-DP 3,6-DPNAc BTC TPB PA POX Fraction H 2,6-DP 3,6-DPNAc BTC TPB PA POX

46 35 19 16 0 . 40 32 30 31 0 .

30

HgC12

30

zero

30

zero

30

98 98 95 75 20

10 0 24 5 9

74 72 75 45 11

.

40 29 47 14 10 .

20 26 15 29 99 100

22 33 15 29 100 100

9 2 0 0 19

76 79 29 9 20

.

45 27 47 12 9 .

9 14 9 9 87 100

15 16 10 10 100 100

97 100 I00 85 0 .

EDTA

zero

99 98 93 78 0 .

Eserine

.

. 95 93 93 85 15

.

.

zero

30

0 0

0 0

-

-

8 100

98 100

0 0

0 0

-

18 2

91 57

Data refer to zero and 30 min inhibition time, and are mean values (two to four runs) obtained on native sera and the respective Fraction II. The enzyme activity in absence of inhibitor was taken as 100% activity (i.e., 0% inhibition).

s. TomiC et al./Biochimica et Biophysica Acta 1251 (1995) 11-16

interesting for its regioselective catalytic properties, was further characterised. Kinetic studies were undertaken with three radiolabelled substrates, 2-MP, 6-MP arid 2,6-DP. Fraction II used in these studies was isolated from one pool of rabbit serum. K M values of 0.95 mM for 2-MP, 1.85 mM for 6-MP and 0.55 mM for 2,6-DP were obtained. Respective values for Vmax (expressed as nmol rain-l m g - i protein) were 3.84 for 2-MP, 28.5 for 6-MP and 140 for 2,6-DP. Optimal pH values for hydrolysis of 2,6-DP catalyzed by enzyme Fraction II was found to be between 7.0 and 10.5. The optimal temperature range for the same substrate was between 30 and 50°C at I0 min reaction time. Specific activities of rabbit serum esterases in native sera and in Fraction II were,, determined using six different substrates, and are presented in Table 1. As compared to native sera, the highest increase in specific activities of Fraction II was observed fi3r the hydrolysis of two sugar substrates, while the increa,;e in activity for BTC and TPB was less pronounced. Phenylacetate and paraoxon, which are substrates of arylesterase and paraoxonase respectively, have a lower specific activity in Fraction II than in native serum. Results on inhibition studies using all the substrates mentioned in Table 1 and five different inhibitors are presented in Table 2. Inhibition of BTC and TPB hydrolysis with HgC12 could not be measured due to interference between the inhibitor and the thiol reagent. When the substrate was paraoxon, we.. used only EDTA and HgC12 as inhibitors.

4. Discussion Carbohydrates represent a challenging target for various selective chemical modifications since they contain many hydroxyl groups of similar chemical reactivity. Enzymatic regioselective acylations [28-33] and deacylations [34-37] of sugar derivatives have involved enzymes from various sources, ranging from commercially available lipases to proteolytic enzymes. Among these enzymes mammalian esterases play a distinctive role, and studies involving regioselective transformations of sugars catalyzed by pig liver esterase [5], mouse liver and serum esterases [7], and esterases from a number of mammalian sera [8] including rabbit serum [8-14] were recently reported. We showed that mammalian sera contain esterases that could be successfully used in regioselective deacylations of various fully or partially acylated monosaccharides. Comparative studies were also done with human serum and sera from various animals [8], and it was shown that rodent sera, under the conditions used, exhibit esterase activities only, while human and ruminant sera show much lower esterase activity parallel with strong transferase activity which causes intramolecular migrations of the ester groups around the sugar ring. Previous results also showed

15

that esterases with specificity for sugar substrates could be isolated from rabbit serum [12], and further used in the preparation of various sugar derivatives in mild reaction conditions and with a high degree of regioselectivity. In this work we present results on characterization of esterases with specificity for carbohydrate substrates isolated from rabbit serum. The inhibition pattern of the 2,6-DP and 3,6-DPNAc hydrolysis resembled most closely the inhibition pattern of BTC hydrolysis. The hydrolysis of all three substrates was progressively inhibited by the organophosphorus compound POX and by the monomethyl carbamate eserine, and all three substrates were inhibited to the same extent by a given concentration of POX or eserine (Table 2). The hydrolysis of TPB was also progressively inhibited by POX and eserine, but to a smaller degree than the other three substrates. Both these inhibitors, POX and eserine, are known as specific time-dependent inhibitors of serine esterases [16]. The hydrolysis of 2,6-DP, 3,6-DPNAc, BTC and TPB was also progressively inhibited by BNPP, which is considered to be a specific inhibitor of carboxylesterases [27]. It was shown earlier that at concentrations of 0.1 mM or less, BNPP inhibited purified preparations of carboxylesterase, but did not inhibit human or horse serum cholinesterase [22]. Inhibition of BTC and TPB by BNPP, described in this paper, confirmed that rabbit serum cholinesterase has some properties which are characteristic of carboxylesterases [22,23]. Consequently, inhibition resuits with POX, eserine and BNPP strongly indicate that the enzyme(s) hydrolysing 2,6-DP and 3,6-DPNAc belong to serine esterases, which group includes the rabbit serum cholinesterase/carboxylesterase enzyme(s). EDTA, at 1.0 mM concentrations, inhibited only slightly the hydrolysis of 2,6-DP, 3,6-DPNAc, BTC and TPB, while the same concentration of EDTA inhibited completely the hydrolysis of POX and PA. HgC12 did not inhibit the hydrolysis of 2,6-DP and 3,6-DPNAc, while the same concentration of HgCI 2 inhibited completely the hydrolysis of POX and PA. These findings exclude paraoxonase and arylesterase as possible enzymes involved in the hydrolysis of acylated monosaccharides. The inhibition pattern of substrate hydrolysis in native rabbit serum was very similar to that in Fraction II. However, the increase in specific activities in Fraction II was higher for the acylated sugar substrates than for either BTC or TPB (Table 1). If the acylated sugar substrates are hydrolysed by cholinesterase/carboxylesterase one would expect the same increase in specific activities for all four substrates. Separation of rabbit serum proteins by gel electrophoresis showed three active bands revealed with BTC as substrate [22,23]. These results and kinetic studies with specific inhibitors revealed the presence of several butyrylcholinesterase isoenzymes in rabbit serum [22,23]. It is possible that only some isoenzymes hydrolysing BTC are active in the hydrolysis of acylated sugar substrates. This would explain the difference in the degree of purifica-

16

S. Torni( et al. / Biochimica et Biophysica Acta 1251 (1995) 11-16

tion in Fraction II of enzyme(s) hydrolysing sugar substrates and those hydrolysing BTC or TPB. Esterase Fraction II was further characterized by its K M and Vmax values using three different ~4C-labelled sugar substrates, and it was shown that the values obtained depend on the substrate used. 2,6-DP was hydrolyzed at fastest rates, followed by the 6-MP, while 2-MP undergoes very slow hydrolysis. These data are in full accord with results reported on regioselectivity of deacylations observed in synthetic studies, proving again that the primary ester group (6-Piv) is hydrolyzed much faster than the secondary one (2-Piv). Optimal pH and temperature ranges were also determined. These data are especially useful in deciding on reaction conditions in synthetic preparations of desired compounds. In conclusion it might be pointed out that rabbit serum contains a serine esterase(s) which is a valuable catalyst in regioselective organic syntheses of various carbohydrates.

Acknowledgements The authors gratefully acknowledge the assistance of E. Pavkovi6, M. Sc. and Mrs. A. Bunti6.

References [1] Whitesides, G.M. and Wong, C.-H. (1985) Angew. Chem. Int. Ed. Engl. 24, 617-638. [2] Davies, H., Green, R., Kelly, D. and Roberts, S. (1989) Biotransformations in Preparative Organic Chemistry, Academic Press, London. [3] Waldmann, H. (1988) Liebigs Ann. Chem., 1175-1180. [4] Waldmann, H. (1988) Tetrahedron Lett. 29, 1131-1134. [5] Waldmann, H. (1991) Kontakte, Darmstadt, 33-54. [6] Drueckhammer, D.G., Hennen, W.J., Pederson, R.L., Barbas, C.F., III, Gautheron, C.M., Krach, T. and Wong, C.-H. (1991) Synthesis, 499-525. [7] Tomi~, S., Tre~ec, A. and Toma~i6, J. (1987) Comp. Biochem. Physiol. 87B, 761-765. [8] Tomi6, S., Sesarti6, Lj. and Toma~i6, J. (1989) Comp. Biochem. Physiol. 92B, 681-684. [9] Tomi~, S., Toma~i6, J., Sesarti~, Lj. and Lade~i6, B. (1987) Carbohydr. Res. 161, 150-155. [10] Ljevakovi6, D., TomiE, S. and Toma~iE, J. (1988) Carbohydr. Res. 182, 197-205. [11] Tomi6, S., Ljevakovi6, D. and Toma~i6, J. (1989) Carbohydr. Res. 188, 222-227. [12] Tomi6, S., Tre~Eec, A., Ljevakovi6, D. and Toma~iE, J. (1991) Carbohydr. Res. 210, 191-198.

[13] Ljevakovi6, D., Tomi~, S. and Toma~i6, J. (1992) Carbohydr. Res. 230, 107-115. [14] Tomi6, S., Ljevakovi6, D. and Toma~i~, J. (1993) Tetrahedron, 10977-10986. [15] Enzyme Nomenclature. Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (1992) Academic Press, San Diego, CA. [16] Aldridge, W.N. and Reiner E. (1972) Enzyme Inhibitors as Substrates. Interaction of Esterases with Esters of Organophosphorus and Carbamic Acids, 328 pp., North-Holland, Amsterdam. [17] Geldmacher-von Mallinckrodt, M. and Diepgen, T.L. (1988) Toxicol. Environ. Chem. 18, 79-196. [18] Enzymes Hydrolysing Organophosphorus Compounds (1989) (Reiner, E., Aldridge, W.N. and Hoskin, F.C.G., eds.), 265 pp., Ellis Horwood, Chichester. [19] Hoskin, F.C.G. (1990) in Squid as Experimental Animals (Gilbert, D.L., Adelman, W.J., Jr. and Arnold, J.M., eds.) pp. 469-480, Plenum Press, New York. [20] Enzymes Interacting with Organophosphorus Compounds, Chemico-Biological Interactions, Special Issue 87 (1993) (Reiner, E. and Lotti, M., guest eds., Johnson, M.K., Simeon V. and Moretto, A., assoc, guest eds., Hodgson, E., consulting ed.), pp. V-XII, Elsevier, Shannon. [21] Reiner, E., Radi6, Z. and Simeon, V. (1989) in Enzymes Hydrolysing Organophosphorus Compounds (Reiner, E., Aldridge, W.N. and Hoskin, F.C.G., eds.), pp. 30-40, Ellis Horwood, Chichester. [22] Simeon, V., Reiner, E., Skrinjari6-Spoljar, M. and Krauthacker, B. (1988) Gen. Pharmacol. 19, 849-853. [23] Main, A.R., McKnelly, S.C. and Burges-Miller, S.K. (1977) Biochem. J. 167, 367-376. [24] Silver, A. (1974) The Biology of Cholinesterases, Frontiers of Biology 36, 596 pp., North-Holland, Amsterdam. [25] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [26] Ellman, G.L., Courtney, K.D., Andres, V. and Featherstone, R.M. (1961) Biochem. Pharm. 7, 88-95. [27] Cain, K., Reiner, E. and Williams D.G. (1983) Biochem. J. 215, 91-99. [28] Therisod, M. and Klibanov, A.M. (1986) J. Amer. Chem. Soc. 108, 5638-5640. [29] Therisod, M. and Klibanov A.M. (1987) J. Am. Chem. Soc. 109, 3977-3981. [30] Hennen, W.J., Sweers, H.M., Wang, Y.-F. and Wong, C.-H. (1988) J. Org. Chem. 53, 4939-4945. [31] Riva, S., Chopineau, J., Kieboom, A.P.G. and Klibanov, A.M. (1988) J. Amer. Chem. Soc. 110, 584-589. [32] Ciuffreda, P., Colombo, D., Rochetti, F. and Toma, L. (1990) J. Org. Chem. 55, 4187-4190. [33] Adelhorst, K., Bjorkling, F., Gadtfredsen, S.E. and Kirk, O. (1990) Synthesis, 112-115. [34] Colombo, D., Ronchetti, F. and Toma, L. (1991) Tetrahedron 47, 103-110. [35] Sweers, H.M. and Wong, C.-H. (1986) J. Am. Chem. Soc. 108, 6421-6422. [36] Uemura, A., Nozaki, K., Yamashita, J.-l. and Yasumoto, M. (1989) Tetrahedron Lett. 30, 3819-3820. [37] Ballesteros, A., Bernabe, M., Cruzado, C., Martin-Lomas, M. and Otero, C. (1989) Tetrahedron 45, 7077-7082.