Planta

Planta I42, 195-201 (1978)

9 by Springer-Verlag 1978

e-Glucosidase from Grape Berries: Partial Purification and Characterization* Angelo Dal Belin Peruffo, Franco Renosto, and Cosimo Pallavicini [stituto di Chimica Agraria, Via Gradenigo, 6, 1-35100 Padova, Italy

Abstract. ~-Glucosidase (c~-D-glucoside glucohydrolase EC 3.2.1.20) was purified approximately 30-fold from grape berries (Vitis vinifera var. Riesling). Besides maltose the enzyme preparation hydrolyzes to a lesser extent maltotriose, isomaltose, and starch. It has a p H o p t i m u m of 5.1 and a molecular weight of a b o u t 100,000. Tris, glycerol, several mono- and disaccharides were tested as inhibitors. The kinetic behavior of ribose, fructose, cellobiose, sucrose, turanose, methylglucopyranoside, Tris, and glycerol was fully investigated. The inhibition studies suggest a Ping-Pong mechanism, with the second substrate concentration being constant, that can be treated as a Uni Bi system. The purified enzyme is stable when stored frozen at - 2 0 ~ C. The grape-berry c~-glucosidase may exist as multiple forms (pI 7.2 and 8.2 respectively), and it does not require ions for its activity.

Key words: ct-Glucosidase -

Grape berries -

Vitis.

Introduction

The ~-glucosidase (EC 3.2.1.20) that catalyzes the hydrolysis of the products of amylase action, namely, maltose and related oligosaccharides, to glucose, is widely distributed in higher plants. In spite of this, the study of plant c~-glucosidases has been neglected compared to that of amylase and phosphorylases, and compared to the c~-glucosidases fi'om m a m m a l i a n tissues, yeast, and fungi. Hutson and Manners (1965) showed that extracts of eleven plant tissues hydrolyze maltose, nigerose, and isomaltose and that the extracts exhibited the highest activity toward the maltose. By continuous * This work was supported by the Consiglio Nazionale delle Ricerche, Roma, Italy

electrophoresis of alfalfa c~-glucosidase, the authors obtained three enzyme fractions that showed significant variations in the relative nigerase: isomaltase activity; and, although homogeneous enzymes were not obtained, they suggested that the ~-glucosidase might exist as isoenzymes. Marshall and Taylor (1971) isolated homogeneous e-glucosidases from sweet corn. The enzymes have an acidic p H optimum (3.1-3.8) and they exist as true isoenzymes, e-Glucosidases showing a similar optimum activity at acidic pH values were also detected in extracts of normal maize, waxy maize, commercial malt (Marshall and Taylor, 1971), and flint corn (Chiba and Shimomura, 1975a). The ~-glucosidase from flint corn showed a broad specificity, although the authors consider the enzyme to be essentially an acid c~-glucosidase with a preferential activity on malto-oligosaccharides to c~-glucans (Chiba and Shimomura, 1975b). An ~-glucosidase that hydrolyzes maltose, isomaltose, panose, and starch, was purified from malted barley (Jorgensen, 1963, 1964). Takahashi et al. (1971) isolated two homogeneous forms of ~-glucosidase from rice seeds. The two enzyme forms have different Km values for maltose, hydrolyze maltotriose and maltotetraose at different rates, and were inhibited differently by Tris and erythrytol. This paper describes some characteristics and the kinetic behavior of a partially purified ct-glucosidase from grape berries.

Materials and Methods

Chemicals and Supplies Sephadex G-200, CM-Sephadex C-50 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden); Maltose (biochemical

0032-0935/78/0195/$01.40

196 grade) was obtained from Merck (Darmstadt, W. Germany), D ( + ) cellobiose, D ( + ) t u r a n o s e , D ( + ) trehalose, D ( + ) raffinose, D ( + ) galactose, L ( + ) arabinose, D ( - ) ribose, 3-0-methyl-e-D-glucopyranoside, o-dianisidine di HCI, were obtained from Sigma Chemical C o m p a n y (S. Louis, Mo., USA). Maltotriose, isomaltose, and methyl-~-D-glucopyranoside were purchased from Koch-Light Laboratories Ltd. (London, England). Thin-layer chromatographic plates silica gel 60 Fas4 were obtained from Merck, and acrylamide and bis-acrylamide from Bio Rad Laboratories (Richmond, California, USA). Glucose oxidase and horseradish peroxidase were purchased from Boehringer (Mannheim, W. Germany). All other materials were reagent grade. All substrates tested were free from impurities as ascertained by thin-layer chromatography (Sica et al., 1971) before their use.

A. Dal Belin Peruffo et aI.: e-Glucosidase from Grape Berries with 800 ml of convex NaCI gradient (0-0.6 M) in the same buffer, and 8 ml fractions were collected. The c~-glucosidase activity eluted between 0.25 0.35 M NaCI.

Sephadex G-200 Fractionation The active fractions eluted from CM-Sephadex were pooled, concentrated to a final volume of about 7 ml in an Amicon ultrafiltration cell with a Diaflo UM-2 membrane, and applied to a 1.6 x 60 cm column of Sephadex G-200, previously equilibrated with standard buffer. Four-ml fi-actions were collected. The c~-glucosidase activity eluted as a single peak at 1.45 Ve of blue dextran. The active fractions were pooled. This solution, which represented approximately a 30-fold purification over the acetone step, was used for all kinetic studies.

Protein Determination Protein was estimated by absorbance at 280 n m or by the Lowry method, using bovine serum albumin as the standard.

Plant Material Mature grapes, Vitis vinifera var. Riesling, from a local vineyard, were washed, packed, and stored according to the procedure of Arnold (1965).

Enzyme Purification Crude Extract. 1500 g of berries (minus seed) were homogenized for 3 min in an Ultraturrax homogenizer with 1200 ml of a solution containing 1.2 M Na-acetate, 1 m M Na 2 E D T A , 12 m M Na-diethyldithiocarbamate, 10 m M cysteine-HCl, and 10% (wt/vol) Carbowax 4000 (polyethylene glycol mol. wt. 4000). Carbowax 4000 has an affinity for tannins and can split tannin-protein complexes (Goldstein and Swain, 1965). Preliminary experiments demonstrated that Carbowax 4000 was essential for the enzyme extraction. After passage through four layers of cheesecloth, the filtrate was centrifuged at 17,000 g for 10 rain. The pellet was discarded. After centrifugation the p H of the crude extract was 5.4.

Acetone Precipitate The crude extract was brought to 100% saturation with solid (NH4)2SO4. After stirring for 16h at 4 ~ the precipitate was collected by centrifugation at 17,000 g for 15 min and then dissolved in 250 ml of 50 m M Na-acetate buffer, pH 5.4 (hereafter this will be referred to as standard buffer). To the solution, refrigerated in an NaCl-ice bath at - 10 ~ C, 3 volumes of cold acetone were slowly added with continuous stirring. After centrifugation at 12,000 g for 10 rain at - 1 0 ~ C, the precipitate obtained was dissolved in the standard buffer and dialyzed overnight against 7 1 of the same buffer.

CM-Sephadex Chromatography The dialyzed solution was applied to a 2.6 x 70 cm column of CMSephadex C-50 equilibrated with standard buffer. After washing the column with 400 ml of standard buffer, the proteins were eluted

c~-Glucosidase Assay The incubation mixture contained 0.1 ml of maltose of desired concentration, prepared in McIlvaine buffer (Dawson et al., 1969), pH 5, 0.3 ml of the same buffer, and 0.1 ml of c~-glucosidase preparation. The reaction was started by the addition of enzyme. The assay mixture was incubated for 30 min at 37 ~ C. The c~-glucosidase activity was determined from the glucose liberated from maltose by the glucose oxidase method (Lloyd and Whelan, 1969). After glucose reagent addition the reaction mixture was incubated for 50 min at 37 ~ C, and the reaction was terminated by adding, with vigorous mixing, 2.5 ml of 5 N HC1. The absorbance at 525 n m was measured against an appropriate blank in a Perkin Elmer double beam spectrophotometer Mod. 124. The As25 was proportional to the glucose concentration up to 50 gg/ 0.5 ml. The incubation time with glucose reagent is sufficient to transform 98% of the glucose produced by the c~-glucosidase activity. Activity measured by this technique was strictly dependent on the a m o u n t of enzyme tested and was linear with time for at least 45 min. One unit of e-glucosidase activity is defined as the a m o u n t of enzyme that catalyzed the hydrolysis of I pmol of maltose per s at 37 ~ C 0akatal). The specific activity is expressed as gkatals/mg protein. In the inhibition studies, to preclude the possibility that the potential inhibitors influenced the enzymes in the glucose oxidase reagent, standard glucose estimations were made in the presence of each of the tested c o m p o u n d s at the highest concentration used: 15 m M ribose was the only potential inhibitor that affected the reagent enzymes.

Polyacrylamide Eiectrofocusing Isoelectric focusing in acrylamide gel was carried out as described by Wrigley (1971) with 2% (wt/vol) of carrier ampholytes (LKB), pH range 3.5-10. Gel length after photopolymerization was 10 cm. Electrofocusing took place at about 4 ~ C; time run, 16 h; potential, 80 V; cathode solution, 0.4% monoethanotamine; anode solution, 0.2% sulfuric acid. The sample used in each course contained about 300 pg of protein. The gels, deprived of carrier ampholytes by washing in 10% trichloroacetic acid, were stained for proteins with 1% Amido Black 10 B in 7.5% acetic acid for 1 h, followed by a destaining period of 24 h in 7.5% acetic acid. c~-glucosidase activity was located in unstained get slices 1.5 m m thick after extracting each disc with 0.4 ml of McIlvaine buffer (pH 5). The extracts were then incubated at 3 7 ~ with 10 m M maltose, and the glucose released was determined by the standard method.

A, Dal Belin Peruffo et al. : e-Glucosidase from Grape Berries

197

Results

Enzyme Purification

2

Table 1 shows the purification of c~-glucosidase. 29-fold purified preparation was determined to no contaminating activities utilizing any of the strates, products, or inhibitors; therefore it was able for all subsequent studies.

The have subsuit-

"~YOGLOBIN

1.5

~ \ -

GLUCONIDASE

1

Estimation of Molecular Weight

~ i

The results of gel filtration runs suggested that grape e-glucosidase behaved as a single component in terms of enzyme activity with an approximate molecular weight of about 100,000 (Fig. 1).

i

i

i

i i i i I

R

R

I

T

I

I I

[

~

i

i

i i i 1i

.o.~eu~,,R w~.G.. (Loo so...) Fig. 1. Molecular weight determination by gel filtration_ A 1-ml sample was applied to a calibrated Sephadex G-200 column (I.6 x 65 cm) equilibrated with 50 m M Na-acetate buffer (pH 5.4). The standards were myoglobin (mol. wt. 17,800), chymotrypsinogen A (tool. wt 25,000)~ aldolase (tool. wt 147,000), and ferritin (mol. wt. 480,000)

Electrofocusing Experiments The gel electrofocusing of the final c~-glucosidase preparation displayed seven protein bands (Fig. 2A). In order to test the activity of our enzyme preparation on starch and to evaluate the presence of amylase as contaminant, some of the gels were cut in 1.5-ram thick discs. Amylase activity was determined by incubating the gel segments in McIlvaine buffer (pH 5) containing 15 mg/ml soluble starch. The liberated reducing sugars were measured with Sumner's reactive (Arnold, 1965). c~-Glucosidase activity was assayed as described in Materials and Methods. Three zones of activity on starch were detected (named 1, 2, and 3 in Fig. 2 B), but only 1 and 2 exhibited e-glucosidase activity (Fig. 2C). Activity was also found only in zones 1 and 2 when the substrates were 10 mM maltotriose, isomaltose, or p-nitrophenyl-c~-D-glucopyranoside.

A

B

C

D

I-

I-

=

I

11

=

I

12

Fig. 2 A - D . Polyacrylamide electrofocusing of partially purified eglucosidase, A Protein pattern of the enzyme preparation; B activity pattern of e-amylase; C activity pattern of c~-glucosidase; D isoenzymatic pattern of e-amylase

Table 1. Purification of e-glucosidase from grape berries" Fraction

Total protein (rag)

Volume (ml)

Units/ml

Total units

Specific activity b

Yield (%)

Purific factor

Crude homogenate ~ Acetone step CM Sephadex SephadexG-200

1020 107 24

2235 242 110 34

0.18 1.2 2.4 5.8

420 290 264 197

0.28 2.46 8.2

100 69 62.8 46.9

1 8.8 29.3

The glucose released by maltose was determined as described in Materials and Methods after protein precipitation with N a O H and ZnSO~ (final concentrations 0.06 M and 0.03 M, respectively) (Bruni et al., 1969). This previous protein precipitation is not necessary for the Sephadex G-200 purified enzyme since the a m o u n t of glucose determined either in the presence or in the absence of protein was the same b Assayed with 10 m M maltose at 37 ~ C p H 5 c The table does not show the specific activity of the crude extract because it is impossible to obtain a reliable estimate of the protein content using the Lowry, Biuret, and Microkjeldhal methods

A. Dal Beiin Peruffo et al. : ct-Glncosidase from Grape Berries

198

Table 2. Kinetic constants of c~-glucosidase from grape berries Substrate

Km (mM)

llX B E

o

Maitose Maltotriose Isomaltose p-Nitrophenylc~-D-glucosidea

50 _> 0

0

I

I

1

2

0.64 0.21 25 0.79

V

Vmax

(gmol of substrate hydrolyzed x mg protein- 1 x min- 1)

(relative to maltose)

0.18 0.08 0.07 0.001

1.0 0.44 0.39 0.005

I

3

4

5

6

7

8

9

pH

Fig. 3. Effect of pH on enzyme activity and stability. Curve A: reaction velocity, 0; curve B: enzyme stability, n. The reaction was carried out in McIlvaine buffer of appropriate pH. Stability measurements were made by preincubating 0.1 ml of the enzyme in 0.3 ml McIlvaine at the indicated pH values for 60 min. Then 0.1 ml of preincubation mixture were transferred to 0.4 ml McI1vaine buffer, pH 5, containing 10 mM maltose, and the reaction was carried out as stated in Materials and Methods (0.4 ml of McIlvaine buffer were sufficient to readjust the pH to 5). The buffers were adjusted to a constant ionic strength by adding the appropriate concentration of KC1. However, preliminary experiments showed that the ionic strength had no effect on either enzyme activity or stability

Enzyme activity on p-nitrophenyl-~-D-glucoside was determined as reported by Lasman (1975)

( L l o y d a n d W h e l a n , 1969). A f t e r i n c u b a t i o n the gels were s t a i n e d for a m y l a s e activity with 1% I 2 - K I solution. T h e results (Fig. 2 D) i n d i c a t e d b a n d s o f activity o n l y w i t h i n z o n e 3 o f F i g u r e 2B. T h e h i s t o c h e m i c a l r e s p o n s e o f a m y l a s e activity was the s a m e w h e n /~limit d e x t r i n ( V a n O n c k e l e n a n d V e r b e e k , 1969) was u s e d as s u b s t r a t e i n s t e a d o f s o l u b l e starch.

e-Glucosidase Stability

v'l

T h e p u r i f i e d e n z y m e was stable at - 2 0 ~ for at least two m o n t h s . O n r e p e a t e d f r e e z i n g a n d t h a w i n g , the e n z y m e s o l u t i o n did n o t s h o w a n y loss of activity. The ~-glucosidase p r e p a r a t i o n r e m a i n e d fully active for m o r e t h a n 1 h at 37 ~ C a n d at p H 5.

3q3

Effect of pH on Enzymatic Activity and Stability

.;

.

;

Fig. 4. Lineweaver-Burk plots of ~-glucosidase. Maltose, 9 ; maltotriose, m. Initial velocity, v, is expressed as gmol of maltose hydrolyzed rain-1 mg protein-t at 37~ C, pH 5. The initial velocity for maltotriose was evaluated assuming that glucose is not produced by the hydrolysis of maltose formed during the reaction (Carter and Smith, 1973)

B o t h e - g l u c o s i d a s e v a r i a n t s were s h o w n to be free f r o m a m y l a s e c o n t a m i n a t i o n b y the f o l l o w i n g experim e n t s : E n t i r e gels were i n c u b a t e d in a r e a c t i o n mixt u r e c o n t a i n i n g 15 m g / m l o f s o l u b l e starch, 0.2 M NaC1, a n d 0.2 M T r i s - a c e t a t e b u f f e r , p H 6.9. Tris is a w e l l - k n o w n i n h i b i t o r o f c~-glucosidase activity

F i g u r e 3 s h o w s the effect o f p H o n e n z y m e activity a n d stability t o w a r d m a l t o s e . M a x i m u m activity o f grape e - g l u c o s i d a s e was o b t a i n e d b e t w e e n p H 5.0 a n d 5.1. T h e e n z y m e was stable for at least 1 h at 37 ~ C b e t w e e n p H 4 a n d p H 7.

Substrate Specificity and Initial Velocity Studies Maltose, maltotriose, isomaltose, p-nitrophenyl-e-Dg l u c o p y r a n o s i d e , a n d s o l u b l e s t a r c h served as s u b strate for c~-glucosidase. N o activity was detected with 1 a n d 10 m M sucrose, trehalose, cellobiose, t u r a n o s e , e - m e t h y l g l u c o s i d e , a n d raffinose. F i g u r e 4 shows the 1/V v e r s u s 1/[maltosaccharide] plots for m a l t o s e a n d m a l t o t r i o s e . The Km a n d V m a x v a l u e s for three m a l t o s a c c h a r i d e s a n d p - n i t r o p h e n y l , - D - g l u c o p y r a n o s i d e are s u m m a r i z e d in T a b l e 2.

199

A. Dal Belin Peruffo et al.: ~-Glucosidase from Grape Berries

/~

(a) 3( (a)

V-I 20

V-1

1 "2

-1

[..,,o,,] ' ..-,

2~-

-20

-10

Ib)

K

0

10

"20

Z~

-10

(c)

0

10

-3

-2

-1

0

..

,.

Fig. 5a-e. Inhibition by sucrose, a Reciprocal of forward velocity vs. 1/[maltose] at the following concentrations of sucrose: 9 e ; 5 mM 9 10 m M zx; 15 mM 9 b Slope replot, e Krn~pv replot

(c) i

(b|

Vm,~

(a) 30

5

V-1

, 0 2o

15 30 [CEL LOBIOSE] m M

- 0.25

,/ 0.25 0.51 ~-ELLOBIOSE]-raM-1

Fig. 7a-e. Inhibition by cellobiose, a Reciprocal of forward velocity vs. 1/[maltose] at the following concentrations of cellobiose; 9 9 ; 2mM 9 8 r a M 9 2 0 r a M 9 3 0 m M []. b I/v-axisintercept replot, e 1/A (1/v-axis intercept) vs. 1/[ceIlobiose] (Segel, I975)

-

~

-60

3

-40

-2

-20

0

-1

20

i 1

0

40

60 ,,,.

i 2

-30

i i J 3 4 5 6 -1 [MALTO, E] mM-1

0

30

i 7

itors. Tris, glycerol, methyl-~-D-glucopyranoside, turanose, and sucrose were linear competitive inhibitors. Fructose and ribose showed linear mixed-type inhibition, whereas cellobiose was a partial inhibitor. Data for sucrose, fructose, and cellobiose are shown in Figures 5 7. Ki and % inhibition are summarized in Table 3. 80 ,,,.

Fig. 6a-c. Inhibition by fructose, a Reciprocal of forward velocity vs. 1/[maltose] at the following concentrations of fructose: 9 9 10 mM 9 ; 20 mM zx ; 30 mM 9 ; 40 mM i . h Slope replot, e 1/vaxis intercept replot

Inhibition Studies Tris-(hydroxymethyl) aminomethane (brought to the desired pH by titration with HC1), glycerol, and a number of saccharides were tested as potential inhib-

Effects of Ions The effect on ct-glucosidase activity of incubation for 30 min at pH 5 with several cations and two anions was tested. While K § Co 2+, Mg 2+, Zn 2+, Cu 2§ Ca 2§ Ni 2+, A13+, C1 , and SO 2- had no effect up to 10 mM, Hg 2§ totally inactivated the enzyme even at 1 mM. Hg 2§ inhibition was completely removed by the addition of disodium E D T A to the reaction mixture.

200

A. Dal Belin Peruffo et al.: c~-Glucosidasefrom Grape Berries

Table 3. Inhibition studies

Inhibitor Tris (hydroxymethyl) aminomethane

mM 2

% of control a

Ki (mM) b

Ki2app (mM) a

80

2

-

Glycerol

150

76

140

-

Ethylene glycol

150

100

-

-

Xylose

10

74

-

-

Arabinose

10

93

-

Galactose

10

100

-

-

3-O-methylc~-glucopyranoside

10

100

-

-

l-O-methylc~-glucopyranoside

10

85

14

-

Fructose

10

72

52 r

25

Ribose

10

62

32.5 c

11.67

Trehalose

10

93

-

-

Turanose

10

5

0.13

--

Sucrose

10

85

18

--

8

64

--

--

Cellobiose

In all experiments 1.5 mM maltose was used as substrate Ki values were determined from the appropriate replots of the primary reciprocal plot data c Kil values determined from slope vs. [I] replot of the primary reciprocal plot data a Ki2"ppvalues determined f r o m 1/Vrnazqpp vs. [I] replot of the primary reciprocal plot data b

Discussion

e - G l u c o s i d a s e o f g r a p e berries is a r a t h e r nonspecific enzyme. It h y d r o l y z e s maltose, m a l t o t r i o s e , isomaltose, a n d p - n i t r o p h e n y l - e - D - g l u c o p y r a n o s i d e . It seems to have a higher " a f f i n i t y " for m a l t o t r i o s e t h a n for m a l t o s e ( K m = 2 X 1 0 4 M a n d 6 x 1 0 - 4 M .respectively) b u t the Vmax for m a l t o s e is a b o u t 2-fold higher t h a n that for maltotriose. This difference m a y be due to the s u b s t r a t e i n h i b i t i o n exhibited by high c o n c e n t r a t i o n s o f the t r i s a c c h a r i d e ( C a r t e r a n d Smith, 1973). The enzyme also exhibits c~-amylase a c t i v i t y like the h o m o g e n e o u s e - g l u c o s i d a s e s f r o m o t h e r sources ( C h i b a a n d S h i m o m u r a , 1975a; Jorgensen, 1964; F l o r e s - C a r r e 6 n a n d R u i z - H e r r e r a , 1972; S i v a k a m i a n d R a d h a k r i s h n a n , 1976). O u r e n z y m e p r e p a r a t i o n is c o n t a m i n a t e d by a - a m y l a s e activity. By gel electrofocusing, however, the two activities can be separated. By the same technique the grape e - g l u c o s i d a s e shows two variants with a pI o f a b o u t 7.2 a n d 8.2, respectively. Thus, the enzyme m a y exist as a mixture o f m u l t i p l e forms similar to the alfalfa ( H u t s o n a n d M a n n e r s , 1965) a n d sweet c o r n ( M a r h s a l l a n d T a y l o r , 1971) e-glucosidases.

The enzyme is c o m p e t i t i v e l y i n h i b i t e d by Tris. Similar i n h i b i t i o n s have been o b s e r v e d for the e-glucosidases o f alfalfa seedlings ( H u t s o n a n d M a n n e r s , 1965), cattle liver (Bruni et al., 1969), r a b b i t muscle ( C a r t e r a n d Smith, 1973; Palmer, 1971), r a b b i t small intestine ( S i v a k a m i a n d R a d h a k r i s h n a n , 1976), a n d b a r l e y m a l t (Jorgensen, 1963; J o r g e n s e n a n d Jorgensen, 1967). J o r g e n s e n a n d J o r g e n s e n (1967) suggested t h a t Tris binds to the enzyme a n d causes a steric h i n d r a n c e for maltose, or changes the charge distribution on the p r o t e i n molecule. The e n z y m e is also c o m p e t i t i v e l y inhibited by glycerol (Ki = 0.14 M), in c o n t r a s t to the results o b t a i n e d with e-glucosidase f r o m o t h e r sources ( C a r t e r a n d Smith, 1973; S i v a k a m i a n d R a d h a k r i s h n a n , 1976; Palmer, 1971). T h e m o n o s a c c h a r i d e s a r a b i n o s e , xylose, galactose, a n d 3 - 0 - m e t h y l - e - D - g l u c o s i d e show little or no inhibition. The e n z y m e is c o m p e t i t i v e l y i n h i b i t e d by emethyl-glucoside. In this respect it differs f r o m o t h e r k n o w n ~-glucosidases ( H u t s o n a n d M a n n e r s , 1965; Palmer, 1971). F r o m the lack o f i n h i b i t i o n by 3 - 0 - m e t h y l - e - D glucoside a n d by galactose, a n d f r o m the i n h i b i t i o n e x h i b i t e d by xylose, it m a y be tentatively c o n c l u d e d that, for the b i n d i n g at the m o n o s a c c h a r i d e i n h i b i t i o n site(s), there is an a b s o l u t e r e q u i r e m e n t for an e q u a t o rial c o n f i g u r a t i o n o f the h y d r o x y l g r o u p at C-3 a n d C-4. The i n h i b i t i o n o f m a l t o s e h y d r o l y s i s by t u r a n o s e was r e p o r t e d as n o n c o m p e t i t i v e ( C a r t e r a n d Smith, 1973), c o m p e t i t i v e (Bruni et al., 1969), a n d mixed type (Jeffrey et al., 1970; Palmer, 1971). In a c c o r d with the results o f Bruni et al. (1969), the grape b e r r y e-glucosidase is c o m p e t i t i v e l y i n h i b i t e d by turanose. Sucrose, r e p o r t e d as n o t an i n h i b i t o r o f c~-glucosidase f r o m r a b b i t muscle (Palmer, 1971), c o m p e t i t i v e l y inhibits o u r enzyme, in a g r e e m e n t with r a b b i t small intestine enzyme ( S i v a k a m i a n d R a d h a k r i s h n a n , 1976). G r a p e c~-glucosidase p r o b a b l y has a P i n g - P o n g r e a c t i o n sequence, as do m a n y o t h e r hydrolases. In the presence o f the fixed c o n c e n t r a t i o n o f water, the m e c h a n i s m can be written as o r d e r e d Uni Bi (Segel, 1975). A = maltose

E EI

x"h

/

l-

P = glucose

(EA=EPQ) I

Q = glucose

1

EQ

EQI [--

E I

A. Dal Belin Peruffo et al.: e-Glucosidase from Grape Berries

In this mechanism, with maltose as the varied substrate, competitive inhibition would result from a sugar (or alcohol) binding only to the free E form of the enzyme, whereas linear mixed-type inhibition would be obtained for sugars that bind to both E and EQ enzyme forms. The partial uncompetitive inhibition pattern of cellobiose may be explained assuming that the disaccharide is acting as an alternative first product (Segel, 1975), i.e., it binds to the EQ form of the enzyme and backs up the reaction via an alternative route leading to an alternative substrate. A

P (EA~-EPQ)

I

(ED~EIQ

Q

~

D

EQ I

E

I = CELLOBIOSE

Because maltotriose (glu-c~-l,4-glu-c~-l,4-glu) is a substrate for grape berry c~-glucosidase (Table 2), the presumed alternative trisaccharide glu-c~-l,4-glu-fl1,4-glu might also be a substrate. Another possible explanation is that cellobiose binds to EQ but does not prevent the further reaction, as shown in the reaction sequence below: P E+A~(EA~EPQ)~EQ~Q+E + + I

K il ]',{ EI

I

$'PI

Ki2 ]'{ EQI~Q+EI

When Kil is very high, the slope effect in the replots is not evident. The exact kinetic constants cannot be determined, because, when maltose is the substrate, P and Q are both glucose and it is not possible to measure the rate of a single product formation. With the exception of Hg 2+, which is a strong inhibitor, all of the remaining cations tested as well as SO 2 and C1- are without effect on grape enzyme. The inhibition of Hg 2+ is consistent with that reported for alfalfa (Hutson and Manners, 1965) and cattle liver (Bruni et al., 1969) c~-glucosidases.

201

References Arnold, W.N. : fl-fructofuranosidase from grape berries. Biochim. Biophys. Acta 110, 134-147 (1965) Bruni, C.B., Auricchio, F., Covelli, I. : Acid e-D-glucosidase glucohydrolase from cattle liver. J. Biol. Chem. 244, 4735-4742 (1969) Carter, J., Smith, E.E. : The substrate specificity of neutral c~-glucosidase from rabbit muscle. Arch. Biochem. Biophys. 155, 82 94 (1973) Chiba, S., Shimomura, T.: Purification and some properties of flint corn ~-glucosidase. Agr. Biol. Chem. 39, 1033-1040 (1975a) Chiba, S., Shimomura, T. : Substrate specificity of flint corn e-glucosidase. Agr. Biol. Chem. 39, 1041 i047 (1975b) Dawson, R.C.M, Elliot, D.C., Elliot, W.H., Jones, K.M.: Data for biochemical research, p. 484, London: Oxford at the Clarendon Press 1969 Flores-Carreon, A., Ruiz-Herrera, J.: Purification and characterization of c~-glucosidase from mucor rouxii. Biochim. Biophys. Acta 258, 496-505 (1972) Goldstein, J.L., Swain, T.: The inhibition of enzymes by tannins. Phytochemistry 4, 185-192 (1965) Hutson, D.H., Manners, D.J. : Studies on carbohydrate-metabolizing enzymes : the hydrolysis of e-glucosides, including nigerose, by extracts of alfalfa and other higher plants. Biochem. J. 94, 783-789 (I965) Jeffrey, P.L., Brown, D.H., Brown, B.I.: Studies of lysosomal c~glucosidase, lI. Kinetics of action of the rat liver enzyme. Biochemistry 9, 1416 1422 (1970) Jorgensen, O.B. : Barley malt c~-glucosidase. II. Studies on the substrate specificity. Acta Chem. Scand. 17, 2471-2478 (1963) Jorgensen, O.B.: Barley malt e-glucosidase. V. Degradation of starch and dextrins. Acta Chem. Scand. 18, 1975-1978 (1964) Jorgensen, B.B., Jorgensen, O.B. : Inhibition of barley malt c~-glucosidase by tris (hydroxymethyl)aminomethane and erythritol. Biochim. Biophys. Acta 146, 167-172 (1967) Lasman, M. : Glucosidase activity in Acanthamoeba palestinensis. The effect of glucose and natural glucosides on e- and fl-glucosidases. J. Protozool. 22, 435-437 (1975) Lloyd, J.B., Whelan, W.J.: An improved method for enzymic determination of glucose in the presence of maltose. Anal. Biochem9 30, 467-469 (1969) Marshall, J.J., Taylor, P.M.: Acid c~-glucosidases from plant sources. Biochem. Biophys. Res. Commun. 42, 173-179 (1971) Palmer, T.N. : The maltase, glncoamylase and transglucosylase activities of acid c~-glucosidase from rabbit muscle. Biochem. J. 124, 713-724 (1971) Segel, I.H.: Enzyme kinetics, pp. 813 818, New York: Wiley 1975 Sica, V., Siani, A,, Bruni, C.B., Auricchio, F. : Maltase, isomaltase and gIucoamylase activities of the acid c~-D-glucoside glucohydrolase isolated from cattle liver. Biochim. Biophys. Acta 242, 422-427 (1971) Sivakami, S., Radhakrishnan, A.N. : Kinetic studies on glucoamylase of rabbit small intestine. Biochem. J. 153, 321-327 (1976) Takahashi, N., Shimomura, T., Chiba, S.: ~-glucosidase in rice. I. Isolation and properties of ~-glucosidase I and ct-glucosidase II. Agr. Biol. Chem. 35, 2015 2024 (1971) Van Onckelen, A., Verbeek, R.: La formation des isozymes de t'c~-amylase durant la germination de I'orge, Planta 88, 255 260 (1969) Wrigley, C.W.: Gel electrofocusing. In: Methods in Enzymology, 22, pp. 559 564. Jakoby, W.B., ed., New York-London: Academic Press 1971

We are grateful to Prof. Irwin H. Segel for his constructive suggestions.

Received 31 March; accepted 12 June 1978