Comp. Biochem. Physiol. Vol. 80B, No. 1, pp. 149-156, 1985 0305-0491/85 $3.00 + 0.00 Printed in Great Britain © 1985 Pergamon Press Ltd

KINETIC STUDIES ON fl-D-FUCOSIDASE OF LITTORINA LITTOREA L.

MARIA J. MELGAR, JOSE A. CABEZAS and PEDRO CALVO Department of Biochemistry, Faculty of Biology, University of Salamanca and C.S.I.C., Salamanca, Spain

(Tel: 923-21-6378)

(Received 8 February 1984)

Abstract--l. The enzyme studied shows ,6-o-fucosidase, fl-o-glucosidase and ,6-o-galactosidase activities, always associated in a single peak in all the chromatographic steps.

2. The enzyme shows, at low and high substrate concentrations, two different K~ and Vm~x, suggesting a sub.~trate-activation model; the highest Vm~/Km values are for ,6-o-fucosidase activity.

3. ,6-o-Fucosides and ,6-o-glucosides completely compete for a common active site in mixed-substrates experiments, while fl-D-galactosides only partially compete with both glycosides. With D-fucose, glucose and galactose as inhibitors, the enzyme shows, at low substrate concentrations, coincident K~ values for ,6-o-fucosidase and ,6-D-glucosidase activities, different from those for fl-o-galactosidase activity. With those inhibitors, the enzyme shows a partial competitive-type inhibition.

4. All the kinetic evidences suggest that this enzyme has two different active sites. 5. At high substrate concentrations some activities are activated by D-fucose, glucose and galactose,

probably in relation with a transglycosylation mechanism.

INTRODUCTION

fl-Galactosidase (fl-D-galactoside galactohydrolase, EC 3.2.1.23) and fl-o-fucosidase (fl-D-fucoside fu- cohydrolase, EC 3.2.1.38) activities are catalyzed by the same enzyme in most of the materials investigated (Cabezas and Vazquez-Pernas, 1969; Llanillo et al., 1977; Walker and Axelrod, 1978; Colas, 1980; Kiss et al., 1981; Rodriguez et al., 1982; Cabezas et al., 1983; Calvo e ta / . , 1983; Chinchetru e ta / . , 1983) but these activities are separated in different enzymes in some molluscs and plants (Levvy and McAllan, 1963; Conchie et al., 1967). On the other hand, fl-O- galactosidase and fl-D-glucosidase (fl-o-glucoside glucohydrolase, EC 3.2.1.21) activities are catalyzed by different enzymes in some materials (Gatt and Rapport, 1966; Conchie et al., 1967; Jungalwala and Robins, 1968; Wallenfels and Weil, 1972; Distler and Jourdian, 1973; Lisman and Hooghwinkel, 1974; Tomino and Meisler, 1975) or by the same enzyme from other sources (Heyworth and Walker, 1962; Llanillo et al., 1977; Grover and Cushley, 1977; Walker and Axelrod, 1978; Colas, 1980; Kiss et aL, 1981; Rodriguez et ak, 1982; Cabezas et al., 1983; Calvo et al., 1983; Chinchetru et al., 1983). However in barley and in the limpet (Conchie et al., 1967) fl-o-fucosidase and fl-D-glucosidase activities are as- sociated in the same enzyme, and both are separated from fl-o-galactosidase activity, that is catalyzed by a different enzyme.

In a first report of the Commission on Enzymes of the International Union of Biochemistry (IUB, 196 l), fl-fucosidase was not present, and its activity was considered to be a side activity of fl-galactosidases. However, fl-fucosidase was classified in 1964 as an independent enzyme (IUB, 1965); it was later deleted in 1972 (IUB, 1973), to be finally reclassified in 1978

*p-NPh-glycosides, p-nitrophenyl-glycosides.

(IUB, 1979). Therefore, it seems interesting to study the kinetics of enzymes showing a well defined fl-o-fucosidase activity.

Since giycosidases act in the catabolism of glyco- lipids and glycosaminoglycans, their absence or deficiency result in metabolic disorders because of the accumulation of the physiological substrate.

Well-characterized enzyme that split fl-D-glucoside and fl-D-galactoside linkages may be also useful as analytical tools for structural studies on glyco- conjugates.

Mollusc glycosidases have special substrate specificities in some cases (Conchi et al., 1967). We have studied here a glycosidase that possesses fl-D- fucosidase, fl-D-glucosidase and fl-D-galactosidase activities with peculiar association and kinetics (being fl-D-fucosidase its main activity) to find out whether the enzyme catalyzes the hydrolysis of the three substrates at the same active site or at different sites. An unexpected activation of the enzyme activities was found with D-fucose, glucose or galactose in some cases, at high substrate concentrations, which sug- gests a transglycosylation mechanism coupled (Pazur et aL, 1958; Disfler and Jourdian, 1973; Holmes and O'Brien, 1979; Calvo et al., 1983; Chinchetru et al., 1983).

MATERIALS AND METHODS

Materials

The digestive glands of the periwinkles, Littorina littorea L., were extracted from living samples, p-NPh-glycosides,* I)-fucose, glucose, galactose, ~-D-galactonolactone, bovine serum albumin, sodium azide and DEAE-cellulose (medium mesh) were obtained from Sigma Chemical Co., St. Louis, MO, USA. Sephadex G-200 and Concanavalin A- Sepharose were purchased from Pharmacia, Uppsala, Swe- den. Agarose-aminocaproyl-fl-D-galactosylamine was ob- tained from P-L Biochemicals, Inc., Milwaukee, WI, U.S.A. Ampholines were from LKB Produkter A.B., Bromma,

149

150 MARIA J. MELGAR et al.

Sweden. General laboratory chemicals were obtained from Probus, Spain. All products were of analytical grade.

Enzyme assays

Glycosidase activities were determined in 0.1 M sodium phosphate/citric acid buffer, pH 5.5, and 1 mM p-NPh- glycoside. After incubation at 37°C, 1.0 ml of 0.2 M Na~CO 3 was added to the reaction mixture (1.0ml). The released p-nitrophenol was measured at 400 nm. All the buffer solutions contained 0.02~ NAN3. A unit of enzyme activity (U) was defined as the amount of enzyme which hydrolyzes 1/~mol of p-NPh-glycoside per minute under the assay conditions. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Determination o f pH optima

The samples were incubated at 37~C with the correspond- ing 1 mM p-NPh-glycoside, in 0.1 M sodium phosphate/ citric acid buffer at pH values ranging between 3.5-7.5.

Enzyme preparation

The digestive glands were homogenized as described previously (Calvo et al., 1978). In ammonium sulphate fractionation the enzyme precipitated from 35 to 65~o saturation• Sephadex G-200 was equilibrated and eluted with 50mM potassium phosphate buffer, pH 5.8. DEAE-cellulose chromatography was carried out in 10 mM potassium phosphate buffer, pH 5.8; the enzyme was eluted with a linear gradient (0-500 mM NaC1). The affinity gel, agarose-aminocaproyl-fl-o-galactosylamine, was equilib- rated with 10mM sodium citrate buffer, pH 5.8; after applying the enzyme solution, the column was washed with 1 M NaC1 in that buffer; the enzyme was eluted with 10 mM 7-D-galactonolactone in the same buffer. Concanavalin A-Sepharose chromatography was carried out in 10mM potassium phosphate buffer, pH 5.8, with 1 M NaCI; the enzyme was eluted with a linear gradient (0-5 M) of man- nose. Preparative isoelectric focusing was performed by the method of Vesterberg (1971) in a l l0ml electrofocusing column (LKB Instruments, Bromma, Sweden). The ampho- lyte concentration was 1~o (w/v), with pH ranges from 3.5 to 5.0 or from 4.0 to 5.0. The run was performed in a sucrose gradient (0-50% w/v) at 500 V for 2 days. Polyacrylamide- gel disc electrophoresis was carried out as previously de- scribed (Calvo et al., 1978).

Determination o f kinetic parameters

The reaction mixtures were incubated at 37°C for 10 min in all the kinetic assays. Michaelis constants (Kin) and maximal velocities (Vm~) were determined from the Lineweaver-Burk plots (1934). The inhibition type was determined from the double-reciprocal and the Dixon plots (1953), with D-fucose, glucose and galactose as competitive inhibitors. Inhibitors constants (K~) were determined by the Dixon plots.

Mixed-substrates analysis

Mutual competition studies between substrates were car- ried out as previously described (Calvo et al., 1983), by using equimolar mixtures of the corresponding p-NPh-glycosides as substrates. The theoretical maximal velocities for com- peting substrates were calculated from the equation applied for competition (Calvo et al., 1978).

RESULTS

Associa t ion of activi t ies

fl-D-Fucosidase, /3-D-glucosidase and /3-D- galactosidase activities, associated in a single peak in Sephadex G-200, DEAE-ce l lu lose and affinity (agarose-aminocaproyl - /3 -D-galactosylamine and

~4

ii N

# g

3i - " °

l

~ a o o

o ¢ tr~ ¢ q r-£

~.~ ~ ~ O ¢ q , . ~ i ~

C b t ' q

o ~ ,~.,~ o 1 ~ . ~ < ~ 1 ~

//-o-Fucosidase of Littorina littorea L. 151

IOO

80

~0

o

~ 20

I I I I I

/, 5 6 7 8

PH

Fig. 1. Effect ofpH on #-D-fucosidase (O), fl-o-glucosidase (D) and #-o-galactosidase (ZX) activities. Experimental

conditions are indicated in the text.

concanavalin A-Sepharose) chromatographies and in 1 pH range isoelectric focusing, are catalyzed by the same enzyme in this material. The purified (more than 400-fold, see Table 1) enzyme preparation shows a single band by polyacrylamide-gel disc electro- phoresis. It does not show any of the other gly- cosidase activities originally present in the crude extract. The activity ratio is constant throughout the purification procedure: fl-D-fucosidase: fl-D- glucosidase: fl-D-galactosidase, 1.00: 0.51: 0.20, under the enzyme-assay conditions. The isoelectric point is 4.25 for all the activities. A single optimal pH was found at pH 5.5 for all the activities (Fig. 1); there- fore, this enzyme does not have the basic and acid forms described by Ho and O'Brien (1971) for

-D-galactosidases from human liver, as we confirmed by the specific assays.

Effect of substrate concentration

The apparent Michaelis constants (Kin) and max- imal velocities (V~,) for each substrate are given in Table 2. Different Km and Vm~ values were found when using low and high substrate concentrations. This suggests a substrate-activation model (Frieden, 1964) discussed later. At low substrate concen-

Table 3. Competition analysis with mixed substrates

V~pt V~,,A + Vm.,S~ V~omp~§ Substrate mixture* (U/mg) (U/mg) (U/rag)

p-NPh-Fuc (A)+ 0.41 1.10 0.47 + p-NPh-GIc (B)

p-NPh-Fuc (A) + 0.63 0.86 0.28 + p-NPh-Gal (B)

p-NPh-GIc (A) + 0.24 0.38 0.18 + p-NPh-Gal (B)

Enzyme activities were assayed as described in the Methods section. *p-NPh-Fuc, p-nitrophenyl-# -D-fucoside; p-NPh-GIc, p-

nitrophenyl-# -D-glucoside; p -NPh-Gal, p -nitrophenyl- #-D-galactoside. Mixed substrates were used at the same concen- tration, ranging from 0.07 to 0.18 raM.

$ ge,p is the experimental Vm,, value obtained from Lineweaver-Burk plots when using mixtures of substrates.

:~V~,,xA + V~,B represents the sum of the maximal velocities ob- tained when the enzyme was separately acting on the corre- sponding substrates A and B.

§ goompt is the theoretical Vm,x calculated from the equation applicable for competing substrates as described in the Methods section.

trations, the enzyme shows the highest Vmax (0.79 U/mg) with p-NPh-fucoside as substrate, and the lowest Km (0.10 mM) with p-NPh-galactoside as substrate (Figs 2, 3). At high substrate concen- trations, the enzyme shows the highest Vmax (0.62 U/mg) and the lowest Km (0.29 mM) with p- NPh-fucoside as substrate (Fig. 4). The ratios Vm~JK,, that indicate the efficacies of several sub- strates used by a single enzyme (Sols and Crane, 1954) are: 3.16ml/mg/min for p-NPh-fucoside, 2.58 for p-NPh-glucoside, and 0.75 for p-NPh- galactoside, at low substrate concentrations; and 2.14 ml/mg/min for p-NPh-fucoside, 1.00 for p-NPh- glucoside, and 0.19 for p-NPh-galactoside, at high substrate concentrations. These results show that fl-o-fucosidase is the main activity of this enzyme, and the galactoside is the worst substrate, at both low and high substrate concentrations.

Competition analysis with mixed substrates

The kinetics of competition with mixed substrates were investigated (Fig. 3) to determine whether this enzyme has one or more active sites for the hydrolysis of fl-D-fucosides, fl-o-glucosides and fl-O- galactosides.

It may be concluded from the results obtained (Table 3) and from the criteria previously indicated

Table 2. Kinetic parameters of the enzyme

Kmt V~xt Fuc Substrate

Glc Gal

*Substrate concentration (mM) (U/rag) (mM) (mM) (raM)

p-NPh-Fuc low 0.25 039 3.75 32.5 175 high 0.29 0.62

p -NPh-GIc low 0.12 0.31 3.75 32.5 175 high 0.39 0.39

p-NPhGal low 0.10 0.075 43.8 87.5 112 high 2.22 0.42

Enzyme activities were assayed under the conditions described in the Methods section. *p -NPh-Fuc, p-nitrophenyl-# -D-fucoside; p -NPh-GIc, p -nitrophenyl-fl-D-glucoside; p-NPh-Gal,

p-nitrophenyl-fl -D-galactoside. tKm and Vm~ ~ values were determined from Lineweaver-Burk plots. The corresponding p-NPh-

glycosides were used as substrates, at concentrations ranging from 0.07 to 0.18 mM (low concentration) and from 1.8 to 3.7 mM (high concentration).

~K i were determined by Dixon plots, with p-NPh-glycosides as substrates at concentrations ranging from 0.07 to 0.16raM, and fucose (Fuc), glucose (Glc) or galactose (Gal) as inhibitors at concentrations ranging from 0 to 200 raM.

152 MARIA J. MELGAR et al.

"E ~ 30 FUC-OS¢uc

-~ /, 8 12 16 ts]-! mM -t

3o

20

10

FUC-as¢ glc

o /

[sl-! mM -1

6O

30 "7 >

- 60 -& E

s 30 >

~ L C - a s ¢

L

8 12 16 -12 [sl-1 mU -I

150 f GAL-as¢

- I00

-8 -/, 4 8 12 16

100

75 OLC-as= / ~ g Ic ~'

50

Is1-! ~M- '

/

J

T

>

J -4

FUC- as.

i

/, B 12 16 -12 -8 -& & 8 12 16 -12 Ls]-', ~ , - ' ~.s]-', ~ , - '

80 GAL - as¢

-~ 60 gal

"7 ~ A0

- 8 ~4 /. a 12 15

Fig. 2. Lineweaver-Burk plots showing the inhibition of fl-D-fucosidase (FUC-ase), fl-D-glucosidase (GLC-ase) and fl-D-galactosidase (GAL-ase) activities, produced by D-fucose (fuc), glucose (glc) and galactose (gal). The inhibitor concentrations were 0 mM (~), 25 mM (1), 50 mM ([7), 100 mM (0) and

200 mM (©). Enzyme activities were assayed as described in the Methods section.

(Calvo et al., 1983) that fl-D-fucosides and fl-D-glucosides completely compete for a common active site: The Vmax obtained with the mixture of both substrates (0.41 U/mg) is almost coincident with the Vma x corresponding to competition (0.47 U/rag), and it is less than one half of the Vmax expected if both activities are catalyzed in separate sites (1.10 U/mg). In contrast, fl-o-galactosidase only partially compete for the common active site for fl-o-fucosides and fl-D-glucosides: The Vma x obtained with the mixtures of the fl-D-galactoside with either the fl-D-fucoside or the fl-D-glucoside (0.63 and 0.24 U/mg) are in be- tween of the Vmax corresponding to competition for an active site (0.28 and 0.18 U/mg) and the Vma~ expected with different active sites (0.86 and 0.38U/mg), suggesting an independent site for fl-D-galactosides. This is also confirmed by the rest of kinetic evidence.

Inhibition kinetics

The inhibition constants (K~) with D-fucose, glucose and galactose were determined considering the lowest inhibitor concentrations used (Fig. 5), that corre- spond to the strongest binding inhibitor-enzyme. The

profiles are not linear in the Dixon plots in most cases, as in the partial competitive-type inhibition (Segel, 1975) discussed later. The Ki values (Table 2) with o-fucose and glucose are significantly higher for fl-o-galactosidase activity (43.8 and 87.5 mM, re- spectively) than the coincident K, values for fl-D-fucosidase and fl-D-glucosidase activities (3.75 and 32.5 mM); in contrast, the Ki value with galactose is lower for fl-o-galactosidase activity (112 mM) than for fl-D-fucosidase and fl-o-glucosidase activities (175 mM), again suggesting a common active site for fl-D-fucosides and fl-o-glucosides and an indepen- dent site for fl-o-galactosides (Dixon and Webb, 1964).

The Lineweaver-Burk plots (Fig. 2) are similar to those corresponding to pure competitive inhibition, while most of the Dixon plots (Fig. 5) are convex upward. This profiles pattern is characteristic for the above-mentioned partial competitive-type inhibition.

Effect of" o-fucose, glucose and galactose on the en- zyme activities, at high substrate concentrations

At high substrate concentrations, fl-o-fucosidase and /~-D-glucosidase activities are unexpectedly acti-

f l -D-Fucosidase of Littorina littorea L. 153

. iI6 I

I I -16

I - 1~6 - 8

~ 2 0 [ Q

_ 'or _ ~ . . ~

- S /, 8 12 16

tO

"7 3o

~" 20

- 8

/.0

i 3O

2O

1 0

[S]~ 1 mM -1

b

t. 8 12 16

IS] -1 ,ram "1

C

I I

, , ;2 ,', [S ] -1 , r a M - 1

Fig. 3. Lineweaver-Burk plots showing the effect of com- peting substrates in the enzyme activities, p-NPh-Fucoside (©), p-NPh-glucoside (F-I) and p-NPh-galactoside (A) were used as substrates, separately (open symbols) or in mixtures (closed symbols): a, p-NPh-fucoside + p-NPh-glucoside (0); b, p-NPh-fucoside+p-NPh-galactoside (&); c, p- NPh-glucoside + p-NPh-galactoside (11). Substrates in mix- tures always had the same concentration. Enzyme activities

were assayed as indicated in the Methods section.

- 3.0 - 2.0 - 1.0 0.5 1.0 [S] - 1 , r a M - |

Fig. 4. Lineweaver-Ourk plots at high substrate concen- trations. (C)), ~-D-Fucosidase activity; (F'q), ~-o-glucosidase activity; (~.), ~-n-galactosidase activity. Enzyme activities

were assayed as described in the text.

DISCUSSION

fl-D-Fucosidase is the main activity of this enzyme, showing with the corresponding p-NPh-glycosides the highest Vm~,/Km value for the ~-t)-fucoside, and the lowest value for the fl-D-galactoside, at both low and high substrate concentrations. Therefore, it can not be considered as a ~-t)-galactosidase with second- ary /~-D-fucosidase activity (Wallenfels and Mal- hotra, 1961; Tomino and Meisler, 1975); in these cases, the fl-D-fucosides are very poor substrates. On the other hand, typical fl-D-galactosidases do not significantly hydrolyze fl-D-glucosides. The enzyme reported here is a real representative of the J~-D-fucosidase entry of the Enzyme Nomenclature (IUB, 1979). The activity pattern of this fl-o-fucosidase with respect to its affinity for the three substrates resembles that of similar enzymes from other snails (Got and Marnay, 1968; Colas, 1980;

vated by D-fucose, glucose or galactose, while fl-D-galactosidase activity is always inhibited (Table 4). fl-D-Fucosidase activity is activated by D-fucoSe and glucose; fl-I)-glucosidase activity is activated by glucose and galactose, but it is inhibited by D-fUcose. Similar results were obtained at other high substrate concentrations not shown in Table 4. All those activities were always inhibited when using low substrate concentrations (Figs 2, 5; Table 2), by competition of the sugars with the corresponding substrates.

These results suggest that the enzyme must have at least two different binding sites for carbohydrates: a modifier site, probably involved in a trans- glycosylation mechanism, that would activate the enzyme when occupied, and another site responsible for the inhibition, that would be the catalytic site(s) where the sugars should compete with the substrates. Those binding sites would be occupied depending on the concentration and different affinity of the sugars for them. Therefore, combinations of activation- inhibition are possible when both sites are occupied.

Table 4. Effect of fucose, glucose and galactose on ~-D-fucosidase, ~-D-glueosidase, and J~-D-galactosidase activities, at high substrate

concentration

mMt Enzyme activity* 0 25 50 100 200

FUC-ase 100 98 115 102 85 Fucose GLC-ase 100 89 74 59 50

GAL-ase 100 70 57 47 39

FUC-ase 100 152 140 122 93 Glucose GLC-ase 100 117 114 98 72

GAL-ase 100 68 59 55 45

FUC-ase 100 95 103 107 108 Galactose GLC-ase 100 105 115 125 126

GAL-ase 100 79 76 70 54

Values given as relative (percents) were calculated from four independent experiments using duplicate samples. Samples incubated without the corresponding monosaccharide were used as controls (100%). p-NPh-Glycosides (2 raM) were used as substrates. The incubation time was l0 rain. Enzyme assays were carried out as described in the Methods section.

*FUC-ase, p-D-fucosidase activity; OLC-ase, p-D-glucosidase ac- tivity; GAL-ase, p-D-galactosidase activity.

tFucose, glucose or galactose concentration.

154 MARIA J. MELGAR et al.

"7" >

-I00

25

-100

FUC-as¢ o fuc /

/

j / ~

, 40 2~0 [ I ] , mM

F U C - o s ¢ g~c

i i i I 100 200

[ I ] , mM

5o

-100

~00 ?~ . . . .

/ /

,;0 2;0 [t], mM

9O

c E 6C

> 30

J -100

GLC - as¢

i I~)0 2100

D] ,~M

-- GAL ese fuc

E f ~

~ / , / l r

I00 I00 200

GAL as¢ [ i ] , m ~ . ~ O L , I00~

~" J ~ v.j j '¢ __~

-100

BO

I0 FUC- ast I "7 20 GLC-ast

-100 100 200 -100 100 200 - 100

[I] , mM rl] , mM

100 200

[i! ,ram GAL ctst gel / / o

f / "

L i , 100 200

D; ,ram

Fig. 5. Dixon plots fl-D-fucosidase (FUC-ase), fl-o-glucosidase (GLC-ase) and fl-D-galactosidase (GAL- ase) activities. D-Fucose (fuc), glucose (glc) and galactose (gal) were used as inhibitors. The concentrations of the corresponding p-NPh-glycosides were 0.068 mM (O), 0.089 mM (O), 0.114 mM (IS]) and 0.160 mM

( I ) . Enzyme activities were assayed as indicated in the Methods section.

Calvo et al., 1983) and from plants (Walker and Axelrod, 1978). From the results obtained in mixed- substrates and inhibition experiments it may be con- cluded that fl-D-fucosidase and fl-o-glucosidase ac- tivities are mainly catalyzed in the fu¢o-g luco site, and ~-o-galactosidase activity, in the galacto site; nevertheless, each activity may be secondarily cata- lyzed in the opposite site.

The enzyme exhibits with D-fucose, glucose or galactose as inhibitors a partial competitive-type inhibition (Segel, 1975) for all the activities, following this scheme:

K~ Kp E + S " . E S - - - + E + P + +

I I

~A', Kp EI + S < . E S I ~ EI + P

The Dixon plots are not linear (Fig. 5); they are convex upward. The velocity of the reactions can never be driven to zero because at infinitely high inhibitor concentration the substrates could always be hydrolyzed in the alternate active site (not oc- cupied by the inhibitor), and both ES and ESI complexes are active. Because both ES and ESI give product with equal facility, the Vm,x will not be

changed. The K, will increase, because at any in- hibitor concentration a portion of the available en- zyme exists in the form EI, having a decreased affinity for S(ctK, > Ks). Therefore, partial competitive in- hibition cannot be distinguished from pure com- petitive inhibition by the corresponding reciprocal plots (Fig. 2). However, it can be distinguished by the Dixon plots (Fig. 5). This mechanism agrees with the two-sites model proposed in the mixed-substrates analysis.

These activities have been reported to be catalyzed in a common active site of the enzyme, in some materials (Llanillo et al., 1977; Colas, 1980). How- ever, in barley and in the limpet (Conchie et al., 1967) fl-o-galactosidase activity is catalyzed by an enzyme different to the one that catalyzes both fl-D-fucosidase and fl-D-glucosidase activities in the same active site. This association of fl-o-fucosidase with//-o-glucosidase activity in a common active site, and not with fl-o-galactosidase activity, is similar to the association of activities in two different active sites, that we describe here. As suggested by Conchie et al. (1967), this unexpected association pattern could be explained by assuming that this glycosidase can have steric hindrance with the substrates, de- pending on the configuration of the glycopyranose ring at C-4 with respect to the group on C-6. As we have found in this enzyme from the mollusc L. littorea, Grover and Cushley (1977) have reported

fl-o-Fucosidase of Littorina littorea L. 155

that fl-D-glucosidase and fl-D-galactosidase activities are catalyzed in two independent active sites, kin- etically distinct, in the fl-D-glucosidase from almond emulsin. With 5-amino-5-deoxy-o-glucose (a gluco- site inhibitor), they also find non-linear, convex up- ward Dixon plots for both activities. On the other hand, Walker and Axelrod (1978) had proposed a single catalytic site for those activities in that ma- terial. However, Kiss et al. (1981) have reported that in that enzyme, fl-D-glucosidase and fl-D- galactosidase activities have two kinetically distinct binding sites in the active center; these results and those previously reported by us in other materials (Caivo et aL, 1983; Chinchetru et al., 1983) show again the occurrence of two independent binding sites for several activities in the same enzyme, as we are reporting here.

All the results reported for mixed-substrate analysis and inhibition kinetics were obtained at low substrate concentrations. When using higher sub- strate concentrations (Fig. 4), the slopes of the Lineweaver-Burk profiles increased with respect to those obtained at low substrate concentrations, deter- mining different Km and Vm~x values, as reported for similar enzymes (Colas, 1980; Lisman and Hoogh- winkel, 1974; Rodriguez et al., 1982). This suggests a substrate-activation model similar to that proposed by Frieden (1964): At high substrate concentration, the enzyme-substrate complex would bind a second substrate molecule, and the resulting substrate- enzyme-substrate complex would yield product at higher velocity. The second substrate molecule can behave as a substrate (SES ~ E + P). The following scheme summarizes the mechanism:

KI K2 E + S . " ES , E + P

K-I +

S

K_3 ]LK~

SES K% E + P

This mechanism could be explained by the presence of a secondary site, that in this case would be the alternate active site (the galacto site for fl-D-fucosidase and fl-o-glucosidase activities and the fuco-gluco site for fl-D-galactosidase activity), in agreement with the two-sites model proposed above.

At low substrate concentration, the substrate would preferentially bind to its primary active site, that would have higher affinity for the substrate, and the enzyme-substrate complex would not bind a second substrate molecule. At high substrate concen- tration, most of the primary active sites are filled, and the substrate binds to its secondary active site, with a lower affinity (K~ increases); then, the complex SES appears, and it yields product at higher velocity (Vm~ increases).

The activation of some activities of this enzyme by o-fucose, glucose and galactose when using high substrate concentrations (Table 4) could be explained by a mechanism of transglycosylation currently as- signed to these enzymes (Pazur et al., 1958; Wall- enfels and Malhotra, 1961). In fact, hydrolysis and transglycosylation are similar mechanisms. The gly-

cosyl residue of the substrate may be transferred either to water or to a glycosyl acceptor. An increase in the acceptor concentration results in enhanced formation of transfer product (WaUenfels and Weil, 1972) and increased release of the aglycone. Regard- less, this activation is unexpected because, at low substrate concentrations, those monosaccharides compete with the substrates in the active sites, in- hibiting all the enzyme activities (Figs 2, 5; Table 2). Therefore, this suggests that, at high substrate con- centrations, more active sites would be occupied with the substrate, and protected from the occupation with the corresponding monosaccharide, so that some monosaccharide could bind to a modifier site (activating the enzyme) and/or to the active sites (competing with the substrates) depending on their affinity and concentration. It has been reported in a related enzyme (Ho, 1973) activation and inhibition by a same modifier, at low and high concentrations of the modifier, respectively, as we have found in some cases (Table 4). The modifier site would be the transfer site for the acceptor; thus, the activation value would depend on the transglycosylation affinities for both the acceptor and the glycosyl residue to be transferred from the substrate. In- hibition should be found when the modifier competes with the substrates.

Acknowledgements--This research has been supported by the Ministerio de Educacirn y Ciencia and by a grant of the Comisi6n Asesora de Investigaci6n Cientifica y T~cnica. The secretarial work by Miss Belrn Blhzquez is acknowl- edged.

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