(Received for publication, April 8, 1996, and in revised form, October 24, 1996)
From the Centre de Recherches sur les
Macromolécules Végétales, ¶ CNRS,
F-38041 Grenoble cedex 9, France and ¶ Novo-Nordisk
a/s, Novo Allé, DK-2880 Bagsvaerd, Denmark
Cellulases are usually classified as
endoglucanases and cellobiohydrolases, but the heterogeneity of
cellulose, in terms of particle size and crystallinity, has always
represented a problem for the biochemical characterization of the
enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate
suitable for measuring cellulase activity by resonance energy transfer
is described. The substrate, which carries a
5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducing
end and an indolethyl group on the reducing end, was prepared from
-lactosyl fluoride and indolethyl
-cellobioside by a
chemoenzymatic approach using the transglycosylating activity of
endoglucanase I of Humicola insolens as the key step. The
bifunctionalized substrate has been used for the determination of the
catalytic constants of H. insolens endoglucanase I and
cellobiohydrolases I and II; this substrate could be of general use to
measure the kinetic constants of cellulases able to act on oligomers of
degree of polymerization <5. The data also provide evidence that
cellobiohydrolases I and II are able to degrade an oligosaccharide
substrate carrying non-carbohydrate substituents at both ends.
Cellulose is a fibrous, insoluble, and crystalline polysaccharide
made of (1,4)-linked D-glucopyranosyl residues and
constitutes the major structural component of plant cell walls and the
most abundant biopolymer on Earth with approximately 109
tons biosynthesized and degraded each year (1). Cellulolytic organisms
(mostly bacteria and fungi) produce a consortium of cellulases usually
classified as cellobiohydrolases (CBH1;
1,4-
-D-glucan cellobiohydrolase, EC 3.2.1.91) and
endoglucanases (EG; 1,4-
-D-glucan glucanohydrolase, EC
3.2.1.4) which degrade cellulose in a synergistic manner (2, 3).
Cellulases are currently grouped in different families, according to a
classification of glycosyl hydrolases based on amino acid sequence
similarities (4, 5). Quite unexpectedly, this classification does not coincide with the above biochemical division since a given family can
contain CBH as well as EG.
Because of the abundance, the properties, and the many uses of cellulose, cellulolytic enzymes have considerable ecological as well as industrial importance. However, the heterogeneity of native cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of cellulases. Small soluble substrates containing a chromophoric group at the reducing end have proved to be very useful for the specificity mapping of various cellulases (6). However, the use of these substrates for kinetic studies is limited since the enzymatic cleavage is monitored directly only when it occurs between the sugar and the aglycon.
The aim of this work is the synthesis of a bifunctionalized
tetrasaccharide substrate for cellulases to provide a sensitive assay
to determine the kinetic parameters of different cellulases using
micromolar concentrations of substrate. This substrate contains two
fluorogenic groups, respectively, at the reducing and non-reducing end
of the molecule. The principle of the assay is based on the fact that
the chromophore donor and the fluorophore acceptor are in sufficient
proximity in the substrate to allow a resonance energy transfer between
the two fluorogenic groups. After hydrolysis of any glycosidic bond in
the substrate, the two fluorogenic groups are separated, and the
resonance energy transfer is no longer possible. The decrease in
resonance energy transfer therefore provides a convenient way to
monitor the enzymatic reaction. This strategy has been used previously
for the assay of proteolytic enzymes (7, 8) and of -amylases (9). To
overcome the problems related to a chemical synthesis of a complex
tetrasaccharide containing four
-1,4-linkages, a chemoenzymatic
approach has been developed for the preparation of the bifunctionalized
substrate. A similar strategy was used recently for obtaining a
disubstituted maltopentaoside for the assay of
-amylases (9). The
enzyme used for the enzymatic condensation is the recombinant
endoglucanase I from the fungus Humicola insolens, which has
previously been shown to exhibit a high transglycosylating activity
(10).
The preparation of the tetrasaccharide indolethyl
6-N-6-(5-(2-aminoethylamino)-1-naphthalenesulfonate)-6-deoxy--D-galactopyranosyl-4-O-(
-D-glucopyranosyl)-4-O-(
-D-glucopyranosyl)-4-O-
-D-glucopyranoside IX, is reported (Fig. 1). This
substrate, which provides a sensitive assay for cellulolytic enzymes,
proved useful for the determination of the kinetic parameters of
several recombinant cellulases from H. insolens.
Enzymes
The H. insolens cellulases were all cloned and
expressed in Aspergillus oryzae (11-13). Endoglucanase V
(EGV) and cellobiohydrolase I (CBHI) were purified using Avicel
affinity chromatography as described (14). EGV and CBHI gave single
bands in SDS-polyacrylamide gel electrophoresis at 43 and 70 kDa,
respectively. Their respective molar absorption coefficients were of
61,300 and 89,700 cm1 M
1.
Endoglucanase I (EGI) and cellobiohydrolase II core (CBHII core) were
purified using ion exchange chromatography. EGI was applied to cation
exchange chromatography using Mono S (Pharmacia Biotech Inc.) and 50 mM sodium acetate buffer, pH 5.0. Pure EGI was eluted using
a pH gradient from 50 mM sodium acetate buffer, pH 5.0, to
100 mM potassium phosphate, pH 7.0. EGI showed a single
band in SDS-polyacrylamide gel electrophoresis at 50 kDa and had a molar absorption coefficient of 66,300 cm
1
M
1. CBHII core was purified by anion exchange
chromatography (DEAE-Sepharose) and 20 mM triethanolamine
buffer, pH 8.0. CBHII core was eluted using a 0-1 M sodium
chloride gradient. The enzyme was purified further by size
chromatography on S-300 Sephacryl (Pharmacia) using 0.1 M
sodium acetate buffer, pH 6.1. Purified CBHII core gave a single band
in SDS-polyacrylamide gel electrophoresis at 42 kDa and had a molar
absorption coefficient of 77,260 cm
1
M
1.
Methods
All of the new compounds displayed elemental analysis and NMR and mass spectra in accordance to their structure. NMR spectra were recorded on a Bruker AC 300 spectrometer at 300 MHz for 1H and 75 MHz for 13C and on a Varian Unity Plus at 500 MHz for 1H. The mass spectra were recorded on a Nermag R-1010C spectrometer. Optical rotations were measured at 20 °C on a Perkin-Elmer 241 polarimeter. The enzymatic condensation reaction was analyzed by HPLC using an analytical NH2 column (µBondapak, Waters) equipped with a refractometric detector and eluted with acetonitrile/water, 77.5:22.5.
Preparation ofHepta-O-acetyllactosyl bromide
I was prepared in a conventional fashion (15). -Lactosyl
fluoride II was synthesized by reacting
hepta-O-acetyllactosyl bromide I (14 g; 0.02 mol)
with silver fluoride (5.2 g; 0.041 mol) in anhydrous acetonitrile (156 ml) for 4 h at 22 °C. The hepta-O-acetyllactosyl
fluoride product was purified by flash chromatography on silica gel
(ethyl acetate/light petroleum, 1:1.5 v/v), crystallized from ether
(yield 63%), and characterized; mp 78-80 °C;
D25 = +9° (c = 1.0 in CHCl3); 1H NMR (300 MHz, CDCl3)
= 5.32 (dd, J(H-11, F) = 53 Hz,
J(H-11, H-21) = 5 Hz,
H-11); 13C NMR (75.4 MHz, CDCl3)
= 170.16-169.00 (7 × CO), 105.55 (d, J(C-11, F) = 218 Hz, C-11), 101.12 (C-12), 20.88-20.34 (7 × CH3). After
deacetylation in the presence of 0.5 M sodium methoxide in
methanol for 3 h at 0 °C,
-lactosyl fluoride II
was neutralized with a (H+) resin, recovered, and used
immediately without further characterization.
Hepta-O-acetylcellobiosyl bromide
III was prepared in a conventional fashion (15).
Glycosylation of bromide III (2.9 g, 4.15 mmol) with
3-(2-hydroxyethyl)indole (333 mg, 2.07 mmol) was achieved in anhydrous
toluene (40 ml) and nitromethane (40 ml) in the presence of
HgCN2 (950 mg) and HgBr2 (67 mg), for 18 h
at 22 °C. The expected disaccharide IV was purified by
flash chromatography on silica gel (ethyl acetate/light petroleum, 1:1
v/v), isolated with a yield of 40% and characterized; mp 96-98 °C; D25 =
20° (c = 0.5 in CHCl3); FAB-MS (3-nitrobenzyl alcohol + KCl): m/z: 779 [M+]; 13C NMR
(75.4 MHz, CDCl3)
= 171.01-168.95 (7 × CO),
136.06, 127.36, 122.24, 121.77, 119.15, 118.52, 112.12, 111.04 (aromatic C in indole), 100.64, 100.54 (C-11,
C-12), 61.88, 61.47 (C-61, C-62),
20.91-20.39 (7 × CH3). After conventional
deacetylation of IV in the presence of 0.5 M
sodium methoxide in methanol, indolethyl cellobioside V was
obtained and characterized;
D25 =
11° (c = 0.5 in H2O); FAB-MS
(glycerol): m/z: 485 [M+];
1H NMR (300 MHz, D2O)
= 7.54 (d,
J = 8 Hz, 1H in indole), 7.36 (d, J = 8 Hz, 1H in indole), 7.11 (m, 1H in indole), 7.10 (s, 1H in indole), 7.02 (m, 1H in indole), 4.33, 4.27 (2d, J = 8 Hz, 2H,
H-11, H-12), 4.05-2.90 (16H); 13C
NMR (75.4 MHz, D2O)
= 137.61, 128.37, 125.00, 123.31, 120.61, 120.08, 113.31, 112.58 (aromatic C in indole), 104.01, 103.56 (C-11, C-12), 62.05, 61.51 (C-61,
C-62).
H. insolens endoglucanase I (134 µl, 4 mg/ml) in maleate buffer (0.05 M, pH 7.0) was added
to a solution of -lactosyl fluoride II (413 mg, 1.2 mmol)
in 4 ml of maleate buffer (0.05 M, pH 7.0) and acetonitrile
(12 ml). After stirring 1 min at 40 °C, indolethyl cellobioside
V (194 mg, 400 µmol) in the same buffer (4 ml) was added.
The solution was kept at 40 °C, and fresh enzyme (134 µl each
time) was added after 10 and 30 min. The reaction was monitored by
HPLC. After 55 min, almost all indolethyl cellobioside V was
consumed, and about 70% of the tetrasaccharide VII was
formed. The solution was brought to pH 9.0 by the addition of
NH4OH, and the mixture was heated for 10 min at 100 °C
to deactivate the enzyme. After a flash chromatography on a reversed
phase column packed with LiChroprep RP-18, 5-20 µm (Merck), eluted
with a gradient from water to water/methanol (70:30 v/v) to eliminate
the buffer and the lactose formed, the mixture was acetylated (24 h) at
ambient temperature with acetic anhydride (6 ml) and pyridine (6 ml) in the presence of a catalytic amount of
N,N-dimethylaminopyridine. After chromatography
on silica gel using ethyl acetate/dichloromethane as eluent (1:3, then
1:2, 1:1, and finally 2:1), the per-O-acetyltetrasaccharide VI (60% yield) was crystallized from ethanol and
characterized; mp 134-137 °C;
D25 =
18° (c = 1 in CHCl3); FAB-MS
(3-nitrobenzyl alcohol): m/z: 1355 [M+]; 13C NMR (75.4 MHz, CDCl3)
= 170.36-169.12 (13 × CO), 136.12, 127.44, 122.24, 121.93, 119.32, 118.64, 112.39, 111.06 (aromatic C in indole), 101.07, 100.61, 100.43, 100.40 (C-11, C-12, C-13,
C-14), 20.83-20.44 (13 × CH3).
Conventional deacetylation of VI afforded pure
tetrasaccharide VII that crystallized from methanol; mp > 280 °C;
D25 =
2°
(c = 1 in H2O); FAB-MS (glycerol):
m/z: 809 [M+]; 1H NMR
(500 MHz, D2O)
= 7.60 (d, J = 8 Hz, 1H
in indole), 7.38 (d, J = 8 Hz, 1H in indole), 7.16 (s,
1H in indole), 7.13 (m, 1H in indole), 7.04 (m, 1H in indole), 4.40, 4.39, 4.38 (3d, J = 7.5 Hz, H-11,
H-12, H-13), 4.27 (d, J = 7.5 Hz, H-14), 4.10-2.95 (28H); 13C NMR (75.4 MHz,
D2O)
= 137.60, 128.28, 125.08, 123.35, 120.60, 120.07, 113.30, 112.60 (aromatic C in indole), 104.39-103.56
(C-11, C-12, C-13,
C-14), 80.03, 79.77, 79.63 (C-41,
C-42, C-43), 62.48 (C-64),
61.47-61.34 (C-61, C-62,
C-63).
Incubation of tetrasaccharide VII (52 mg, 64 µmol) with galactose oxidase from Dactylium
dendroides (Fluka, 36 units) and catalase from bovine liver
(Sigma, 360 units) at 25 °C in 1 mM CuSO4
for 15 h under ~2 bar air pressure gave compound VIII in quantitative yield. Reductive amination of VIII (22.6 mg,
28 µmol) in the presence of EDANS (31.4 mg, 109 µmol) and NaBH3CN (12.5 mg, 199 µmol) in methanol (6 ml) at
22 °C for 24 h gave tetrasaccharide IX. Purification
by chromatography on a reversed phase column packed with LiChroprep
RP-18, 5-20 µm (Merck) eluted first with water and then with a
gradient of water and methanol afforded the bifunctionalized
tetrasaccharide IX (60% yield);
D25 = ~0° (c = 0.23 in dimethyl sulfoxide); FAB-MS (glycerol):
m/z: 1,079 [M+]; 1H NMR
(500 MHz, D2O)
= 8.10 (d, J = 9 Hz, 1H
in EDANS), 8.01 (d, J = 7.5 Hz, 1H in EDANS), 7.96 (d,
J = 9 Hz, 1H in EDANS), 7.60 (d, J = 8 Hz, 1H in indole), 7.46 (m, 1H in EDANS), 7.43 (m, 1H in EDANS), 7.38 (d, J = 8 Hz, 1H in indole), 7.16 (s, 1H in indole),
7.13 (m, 1H in indole), 7.04 (m, 1H in indole), 6.75 (d,
J = 7.5 Hz, 1H in EDANS), 4.40, 4.38, 4.32, 4.30 (4d,
J = 8 Hz, H-11, H-12,
H-13, H-14), 4.10-2.95 (32H); 13C
NMR (75.4 MHz, D2O/CD3OD)
= 145.40, 130.84, 129.38, 128.46, 126.07, 124.65, 116.65, 107.31 (aromatic C in EDANS),
137.67, 127.37, 124.65, 122.95, 120.21, 119.73, 112.89, 112.43 (aromatic C in indole), 104.17-103.72 (C-11,
C-12, C-13, C-14), 61.48-61.33
(C-61, C-62, C-63).
The initial rates of the enzymatic hydrolyses of bifunctionalized substrate IX were performed at 37 °C in 0.1 M sodium acetate buffer, pH 5.0. Reactions were initiated by the addition of enzyme. Initial rate measurements were made at several substrate concentrations, which ranged from 0.1 to 4 times the value of the Km ultimately determined. Values of Km and kcat were determined using Lineweaver-Burk plots and were the result of three determinations. All kinetic studies were monitored with a Perkin-Elmer LS50 spectrofluorometer using an excitation wavelength set at 290 nm and an emission wavelength set at 490 nm.
During the
hydrolysis of oligosaccharides and glycosides under thermodynamically
controlled conditions, certain retaining glycosyl hydrolases such as
-galactosidase (16),
-glucosidase (17), and
-xylosidase (18)
display transglycosylating activity and produce mixtures of higher
oligosaccharides in moderate yields and low linkage specificity. Under
kinetically controlled conditions, more efficient transglycosylation
reactions can be achieved when an activated carbohydrate donor is
incubated with a polysaccharidase. In this case, the resulting higher
oligosaccharides also exhibit good linkage specificity. This approach
was first used for the high yield preparation of malto-oligosaccharides
using maltosyl fluoride and
-amylase (19). This strategy has since
been used for the preparation of natural or non-natural
oligosaccharides using, for instance, amylases (9) or cellulases (20,
21).
Endoglucanase I of H. insolens was chosen for the present enzymatic synthesis because it had been shown previously to exhibit an intense transglycosylating activity using cellotetraitol as substrate, which resulted in the production of insoluble higher cello-oligosaccharides (10).
The present enzymatic synthesis (Fig. 1) was based on the
transglycosylating activity of H. insolens endoglucanase I,
with the following additional constraints: (i) the coupling of two distinct molecules (lactosyl fluoride II and indolethyl cellobioside V) instead of a polycondensation of a substrate on itself; (ii) the selective preparation of a -1,4-tetrasaccharide with minimal production of higher oligomers; and (iii) the subsequent bifunctionalization of the substrate. These constraints explain our
choice of the
-lactosyl fluoride II as the glycosyl donor
in the transglycosylation reaction instead of
-cellobiosyl fluoride,
for example. The ability of cellulases to use
-lactosyl fluoride
II as a substrate has already been demonstrated (22). In
fact, the galactosyl unit of lactose provides two crucial advantages.
First, it prevents a higher polymerization during the condensation
since oligosaccharides with axial 4-OH at the non-reducing end cannot
act as acceptors in the transglycosylation reaction catalyzed by a
cellulase. This enables the single addition of a single lactosyl moiety
onto indolethyl
-cellobioside V. Second, the galactosyl
residue at the non-reducing end of the resulting tetrasaccharide
represents a convenient site for the introduction of the second
fluorescent group via the regioselective oxidation of the C-6 position
by galactose oxidase and subsequent reductive amination.
The principle of the transglycosylation reaction is as follows. The
enzyme initially reacts with the only substrate available, i.e. lactosyl fluoride II, with the formation of
a lactosyl-enzyme intermediate and liberation of hydrogen fluoride.
Then the lactosyl unit is transferred to the acceptor, indolethyl
cellobioside V, yielding tetrasaccharide VI. In
this scheme, the major competing reaction is the simple hydrolysis of
the sugar donor and of the sugar acceptor. High substrate
concentrations usually favor transglycosylation reactions. In some
cases, the addition of an organic solvent to the reaction medium can
help transglycosylation. In fact, early experiments involving the
incubation of H. insolens endoglucanase I with -lactosyl
fluoride II and indolethyl
-cellobioside V in
buffer only resulted in the hydrolysis of both donor and acceptor (data
not shown). Various organic solvents have thus been assayed to find
good conditions for the transglycosylation reaction. With the present
system, we found that a 1.5:1 (v/v) mixture of acetonitrile and maleate
buffer (0.05 M, pH 7.0) gave good results. Although the
presence of an organic solvent appears necessary for the
transglycosylation, these conditions are detrimental for the activity
of the enzyme, which showed progressive denaturation after 15-20 min.
The problem was circumvented by the addition of fresh enzyme during the
reaction. Finally, various donor/acceptor concentrations have been
explored, and the best results were obtained with donor and acceptor
concentrations of 60 and 20 mM, respectively. Using the
above conditions, the monofunctionalized tetrasaccharide VI
was isolated with a yield of 60%. Small proportions of other
oligosaccharides deriving from side reactions were also readily
isolated (Table I). The bifunctionalization of
tetrasaccharide VII was achieved by the oxidation of the C-6
of the terminal galactose unit by galactose oxidase followed by
reductive amination. The bifluorescent tetrasaccharide IX
was thus obtained with a yield of 60% from tetrasaccharide
VII and an overall yield of 14% from cellobiose
octaacetate. The 1H NMR spectrum of tetrasaccharide
IX is shown in Fig. 2.
|
Kinetic Assays
The efficiency of intramolecular resonance
energy transfer depends on the distance between donor and acceptor and
on the spectral overlap between the donor emission and the acceptor
absorption (23). Among the different donor-acceptor pairs potentially
useful for energy transfer measurements, the pair chosen in our study was indolethanol-EDANS, for which the efficiency of the resonance energy transfer appeared satisfactory. Indolethanol absorbs at max = 290 nm and emits at
max = 365 nm;
EDANS absorbs at
max = 340 nm and emits at
max = 490 nm (Fig. 3). In addition, these two chromophoric groups can be linked to oligosaccharides via stable
bonds (9).
The first important experiment was to verify the intramolecular
resonance energy transfer. When the bifunctionalized tetrasaccharide IX (5-20 µM) was excited at 290 nm, the EDANS
fluorescence recorded at 490 nm was five times higher than that of a
solution of free EDANS at the same concentration. On the other hand,
the resonance energy transfer was found to decrease the fluorescence of
the indolethyl moiety at 365 nm by 50% compared with the fluorescence of the monofunctionalized tetrasaccharide VII (Fig.
4). These values, which demonstrated the resonance
energy transfer, also indicated that enzymatic hydrolyses would be
monitored more sensitively by the decrease of the fluorescence at 490 nm than by the increase of the fluorescence at 365 nm.
The substrate concentrations used must be sufficiently low to prevent
intermolecular resonance energy transfer. The fluorescence intensity at
490 nm of a 20 µM solution of free EDANS was found to be
almost identical to that of a solution containing EDANS and indolethyl
tetrasaccharide VII at the same concentration, demonstrating
that intermolecular transfer can be neglected at this concentration.
The kinetic assays have therefore been performed with substrate
concentrations ranging from 2 to 20 µM. Recording the
fluorescence intensity of the bifunctionalized tetrasaccharide IX at different concentrations (5, 10, 15, and 20 µM) for 1 min demonstrated that the fluorescence
intensity at 490 nm is proportional to the substrate concentration. The
decrease in fluorescence observed during enzymatic hydrolysis (an
example is given in Fig. 5) is therefore directly
related to the rate of hydrolysis since for each mol of substrate which
is cleaved, the total fluorescence is decreased by the intensity caused
by 1 mol of EDANS. Initial rates and therefore kinetic parameters are
directly available from the slope of the straight lines recorded.
Four fungal recombinant cellulases from the fungus H. insolens have been tested for their ability to hydrolyze the bifunctionalized tetrasaccharide IX. These enzymes were two endoglucanases, EGI and EGV, and two cellobiohydrolases, CBHI and CBHII (10). The values of Km and kcat obtained for the various enzymes are presented in Table II.
|
EGV is the only enzyme with which no hydrolysis was detected. This
result is compatible with earlier observations that EGV contains at
least six subsites and that the catalytic constants for the hydrolysis
of cellotetraitol are very poor with a Km above
1,000 µM and a kcat = 0.84 s1 (10). On the other hand, hydrolysis of cellohexaitol
by EGV was much more efficient (Km = 52 µM and kcat = 14 s
1)
(10). The bifunctionalized tetrasaccharide IX is probably too short, and/or the chromophoric groups perhaps prevent the productive binding of the substrate to the enzyme.
The second enzyme tested was EGI, the enzyme used for the enzymatic
synthesis of the substrate. EGI proved to be very efficient for the
hydrolysis of the bifluorescent substrate IX with values of
kcat (27 s1) and
kcat/Km (3.4 s
1
µM
1) comparable to the values of 40 s
1 and 2.9 s
1
µM
1), respectively, obtained with the same
enzyme using cellopentaitol as substrate (10). This observation shows
that the large fluorogenic groups introduced on the tetrasaccharide
have little influence on the hydrolysis of the substrate by this
enzyme.
Surprisingly, the two cellobiohydrolases, CBHI and CBHII core, were
also able to hydrolyze the bifunctionalized substrate with
Km values in the micromolar range (Table II). The kinetic parameters show, however, that these two enzymes are less efficient than EGI since their catalytic constants were found to be,
respectively, 0.40 and 0.023 s1 (Table II). The value of
0.40 s
1 obtained with CBHI, albeit low, is comparable to
the value of 0.96 s
1 obtained during the hydrolysis of
cellopentaitol (10). This result indicates that the two fluorogenic
groups of the substrate do not affect very significantly hydrolysis of
the substrate by CBHI. CBHII core is also able to cleave the
bifunctionalized tetrasaccharide, although with a catalytic constant
largely reduced compared with that observed against cellopentaitol
(0.67 s
1) (10). The sites of cleavage of the
bifunctionalized substrate by EGI, CBHI, and CBHII were analyzed by
thin layer chromatography (data not shown). EGI and CBHI were found to
be able to hydrolyze the substrate at two points, whereas CBHII could
cleave only one of the interosidic bonds (Fig. 6).
CBHI and CBHII from H. insolens display high sequence similarity with CBHI and CBHII from Trichoderma reesei (63 and 58% sequence identity, respectively), whose three-dimensional structures have been determined (24, 25). The only feature common to these otherwise dissimilar protein structures is the topology of their active sites. In both cases, long loops cover part of the active site cleft, and the resulting tunnel enables the threading of a cellulose chain (25). The tunnel-shaped active site of cellobiohydrolases permits the liberation of the cellobiose product while maintaining the cellulose chain bound to the enzyme. This active site topology allows the processive, recurrent hydrolysis of cellulose chains into cellobiose (26). An important question remains with the site of the initial attack of cellulose. Three possibilities exist: (i) entry of the substrate chain end from only one entrance of the tunnel resulting in a selectivity for a single chain end type; (ii) entry of reducing chain ends from one side of the tunnel while non-reducing chain ends could enter from the other side; and (iii) a sporadic opening of the loops to allow binding of a cellulose chain in the active site. Current models favor the first possibility with a selective chain end attack for cellobiohydrolases, with CBHI starting from the reducing ends (27, 28) and CBHII starting from the non-reducing ends of cellulose chains (28, 29). The fact that the two recombinant cellobiohydrolases we have assayed are able to degrade the bifunctionalized substrate carrying non-carbohydrate substituents at both ends argues against the specific chain end recognition by cellobiohydrolases and suggests that a different model for the initial attack should be envisioned.
In conclusion, the use of the transglycosylating activity of the
recombinant endoglucanase EGI from H. insolens has provided a rapid and efficient synthesis of a bifunctionalized tetrasaccharide from two modified disaccharides. This chemoenzymatic approach has led
to the preparation of a useful substrate for the biochemical characterization of cellulases since it provides a very sensitive assay
that can be monitored continuously. Furthermore, the comparison of
catalytic constants with those obtained on reduced cellodextrins shows
that the fluorophores do not seem to influence the enzymatic hydrolysis
by cellulases having a low number (5) of subsites. For cellulases
such as H. insolens EGV which have a large number of
subsites (>5), the synthesis of a longer oligosaccharide should be
envisaged. Since the efficiency of the resonance energy transfer depends both on the distance between the two chromophores and on the
spectral overlap of donor and acceptor, the use of another donor-acceptor pair that would show a better spectral overlap should be
considered, to compensate for the increased distance between the two
fluorophores when one or two extra glucosyl residues are added. In
addition to providing a sensitive assay for endoglucanases, the
bifunctionalized tetrasaccharide was also found to be hydrolyzed by two
cellobiohydrolases, suggesting that these enzymes do not specifically
require a free chain end for their action. Work is currently under way
in our laboratory to test this hypothesis further.
We are grateful to Drs. S. Cottaz (CERMAV) and G. Davies (University of York) for useful discussions. We also thank C. Gey (CERMAV) for performing the NMR and Dr. C. Bosso (CERMAV) for performing the FAB-MS.