Mechanism of the action of Leuconostoc mesenteroides B-512FMC dextransucrase: Kinetics of the transfer of D-glucose to maltose and the effects of enzyme and substrate concentrations

Article abstract of DOI:10.1016/S0008-6215(99)00155-X

The kinetics of the reaction of Leuconostoc mesenteroides B-512FMC dextransucrase with sucrose were studied. This enzyme catalyzes the synthesis of dextran from sucrose with a k(cat) of 641 s^-1 and the transfer of D-glucose from sucrose to maltose with a k(cat) of 1070 s^-1. The enzyme was also found to catalyze two new reactions in the absence of sucrose, using dextran as the substrate; D-glucose was transferred from the non-reducing ends of dextran chains to maltose with a relatively low k(cat) of 3.2 s^-1; and D-glucose was hydrolyzed from the non-reducing ends of dextran chains with a very low k(cat) of 0.085 s^-1. Ping-pong/bi-bi kinetics of these reactions are consistent with the formation of a glucosyl-enzyme covalent intermediate. It is shown that an increase in the concentrations of both maltose and sucrose in the D-glucose transfer reaction to maltose gives an exponential decrease in the amount of dextran and a concomitant increase in the amount of acceptor products. It is further shown that increasing the amount of dextransucrase gives a decrease in the amount of dextran and an increase in the amount of acceptor products, after the sucrose has been consumed. This anomaly occurs because the relatively high amounts of enzyme catalyze the transfer of D-glucose from the non-reducing ends of the dextran chains to maltose, giving a decrease in the amount of dextran and an increase in the amount of acceptor product. Further, the high amounts of enzyme catalyze the hydrolysis of the D-glucose residues from the ends of the dextran chains, giving a decrease in the amount of dextran. These reactions are not observed when lower amounts of enzyme are used, as the reactions are much slower than the synthesis of dextran and the usual acceptor transfer reactions of D-glucose from sucrose to acceptor. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00155-X

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Carbohydrate Research 320 (1999) 183–191
Mechanism of the action of Leuconostoc mesenteroides
B-512FMC dextransucrase: kinetics of the transfer of
D-glucose to maltose and the effects of enzyme and
substrate concentrations
Motomitsu Kitaoka 1, John F. Robyt *
Laboratory of Carbohydrate Chemistry and Enzymology, 4252 Molecular Biology Building, Iowa State Uni6ersity,
Ames, IA 50011, USA
Received 21 January 1999; accepted 20 May 1999
Abstract
The kinetics of the reaction of Leuconostoc mesenteroides B-512FMC dextransucrase with sucrose were studied.
This enzyme catalyzes the synthesis of dextran from sucrose with a kcat of 641 s−1 and the transfer of
D
-glucose from
sucrose to maltose with a kcat of 1070 s−1. The enzyme was also found to catalyze two new reactions in the absence
of sucrose, using dextran as the substrate; -glucose was transferred from the non-reducing ends of dextran chains
D
to maltose with a relatively low kcat of 3.2 s−1; and
D-glucose was hydrolyzed from the non-reducing ends of dextran
chains with a very low kcat of 0.085 s−1. Ping-pong/bi-bi kinetics of these reactions are consistent with the formation
of a glucosyl–enzyme covalent intermediate. It is shown that an increase in the concentrations of both maltose and
sucrose in the
D-glucose transfer reaction to maltose gives an exponential decrease in the amount of dextran and a
concomitant increase in the amount of acceptor products. It is further shown that increasing the amount of
dextransucrase gives a decrease in the amount of dextran and an increase in the amount of acceptor products, after
the sucrose has been consumed. This anomaly occurs because the relatively high amounts of enzyme catalyze the
transfer of
of dextran and an increase in the amount of acceptor product. Further, the high amounts of enzyme catalyze the
hydrolysis of the -glucose residues from the ends of the dextran chains, giving a decrease in the amount of dextran.
These reactions are not observed when lower amounts of enzyme are used, as the reactions are much slower than the
D-glucose from the non-reducing ends of the dextran chains to maltose, giving a decrease in the amount
D
synthesis of dextran and the usual acceptor transfer reactions of
Science Ltd. All rights reserved.
D-glucose from sucrose to acceptor. © 1999 Elsevier
Keywords: Dextransucrase; Maltose acceptor reactions; Transfer reactions; Hydrolytic reactions; Effects of substrate concentra-
tions; Effects of enzyme concentration; Kinetics; Leuconostoc mesenteroides B-512FMC
1. Introduction
It is generally recognized that dextransu-
crase catalyzes three reactions, using sucrose
as the substrate: (1) the synthesis of dextran,
(2) the synthesis of dextran branch linkages,
and (3) in the presence of other carbohydrates,
* Corresponding author. Tel.: +1-515-294-6116; fax: +1-
515-294-0453.
E-mail address: jrobyt@iastate.edu (J.F. Robyt)
1 Present address: National Food Research Institute, Kan-
nondai, Tsukuba, Ibaraki 305, Japan.
such as
D
-glucose,
D-fructose, maltose, etc.,
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00155-X

184
M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
studied, along with the effects of the substrate-
the transfer of
D
-glucose from sucrose to the
and the enzyme concentrations on the accep-
tor reaction in an attempt to understand the
effects of substrate and enzyme concentrations
on the reactions.
carbohydrate (reviewed in Ref. [1]). The latter
two reactions are commonly called acceptor
reactions.
The mechanism of the synthesis of dextran
has been shown to involve a two-site insertion
mechanism in which
ing dextran chain are covalently attached to
the active site. The -glucose is transferred to
D
-glucose and the grow-
2. Experimental
D
Materials
the reducing end of the growing dextran chain
[1,2]. The branch linkages by the action of the
synthetic enzyme (dextransucrase) form when
a dextran chain acts as an acceptor and dis-
Enzyme. Dextransucrase was prepared by
growing a high-producing constitutive mutant
of L. mesenteroides B512FMC-16 [6] in a
D
-
glucose medium and purifying it as previously
described [7]. The enzyme used in this report
was electrophoretically pure on PAGE, with a
molecular mass of 190 kDa. The dextransu-
crase activity was measured using [U-
14C]sucrose as previously described [6]. One
international unit (IU) of dextransucrase ac-
tivity is defined as the amount of enzyme that
places either the
D
-glucose or the dextran
chain from the active site, forming a-(1“3)
branch linkages [1,3].
Leuconostoc mesenteroides B-512F dextran-
sucrase can transfer -glucose from sucrose to
D
several different carbohydrate acceptors [1,4].
Maltose is known to be the most effective
acceptor [4], forming panose [62-a-
-glucopy-
D
incorporates 1 mmole of 14C-
-glucose into
D
ranosyl maltose], which also can be an accep-
tor to give a tetrasaccharide, which also can
be an acceptor to eventually give a ho-
mologous series of 62-a-isomaltodextrinyl mal-
tose products [4,5]. Su and Robyt [5] studied
the effects of the ratio of maltose to sucrose
(M/S) on the acceptor reaction and found that
the ratio significantly affected the amount and
number of products formed. When the ratio
dextran from 125 mM [U-14C]sucrose at
21 °C. The specific activity of dextransucrase
was 183 IU mg−1
.
Dextrans. B-512F-Dextran T-10, T-40, and
T-500 were obtained from Pharmacia Biotech
Inc. (Piscataway, NJ). The properties of the
dextrans are summarized in Table 1.
Methods
Inhibition of the synthesis of dextran by mal-
tose. All dextransucrase reactions contained 1
mg mL−1 Tween 80 and 1 mM CaCl2 to give
maximum activity and enzyme stability [8,9].
A series of reactions was carried out in 200
mL digests containing 1.3 mCi mL−1 [U-14C]-
sucrose at concentrations of 5, 10, 20, 30, or
50 mM and maltose concentrations of 0, 20,
or 40 mM. The reactions were initiated by the
addition of 0.05 IU of dextransucrase per mL,
which was in 10 mM NaOAc buffer (pH 5.2)
containing 1 mg mL−1 Tween 80 and 1 mM
CaCl2 at 21 °C. The amount of radioactivity
incorporated into dextran was measured every
10 min for 40 min by taking 25 mL aliquots
and adding them to 1.5×1.5 cm Whatman
3MM filter papers, which were immediately
dropped into MeOH; the papers were washed
twice with MeOH, and the amount of 14C
present on the paper was determined in a
toluene cocktail by liquid scintillation
spectrometry.
was low (0.2), about 50% of the
D
-glucose
available from sucrose was incorporated into
dextran and the remaining 50% was incorpo-
rated into eight maltose acceptor-products.
When the maltose to sucrose ratio was high,
for example when M/S=100 (100 mM mal-
tose+1 mM sucrose) and when M/S=20 (2
M maltose+100 mM sucrose), no dextran
was formed and only a single trisaccharide
product, panose, was formed [5]. Su and
Robyt [5] also found that the distribution of
D-glucose into dextran and into the acceptor
products was dependent on the concentrations
of sucrose and the acceptor. Further, they
found that the increase in the amount of
enzyme caused a decrease in the amount of
dextran and a concomitant increase in the
amount of acceptor products. These two ob-
servations were relatively unusual and not
readily explained. In the present study, the
kinetics of the maltose acceptor reaction were

M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
185
and 1 mM CaCl2 with 0.125 IU mL−1 dex-
transucrase at 21 °C.
Effect of the enzyme concentration on the
maltose acceptor reaction. The maltose accep-
tor reaction, conducted by using different
amounts of enzyme, was performed in 400 mL
digests containing 5 mCi mL−1 [U-14C]sucrose
(12.5 or 50 mM) and dextransucrase (42, 4.2
or 0.42 IU mL−1) in 10 mM NaOAc buffer
(pH 5.2), containing 1 g L−1 Tween 80 and 1
mM CaCl2 at 21 °C. Aliquots (25 mL) of the
digests were added to 5 mL of 60% (v/v)
pyridine at various times to make the pH 10
and stop the reaction. Then, 10 mL of the
solution was spotted onto a Whatman K6F
TLC plate (20×20 cm), followed by three
ascents with 7:3 (v/v) MeCN–water, path-
length of 18.5 cm. The labeled compounds,
The
D-glucosyl transfer reaction from dex-
tran to maltose was studied, using three kinds
of dextrans (dextran T-10, T-40, and T-500) at
concentrations of 1, 2, 5, 10 mg mL−1 and 50
mM maltose in 10 mM NaOAc buffer (pH
5.2), containing 1 mg mL−1 Tween 80 and 1
mM CaCl2 with 12.5 IU mL−1 dextransucrase
at 21 °C. The kinetic constants of each dex-
tran as a glucosyl donor were determined. The
mechanism of the reaction was further deter-
mined by using various concentrations of
1, 2, 4, and 10 mg mL−1 dextran T-500 with
20, 40, and 80 mM maltose.
To measure the increase in the acceptor
product (panose), 50 mL-aliquots of the di-
gests were taken every 10 min for 40 min and
added to 50 mL of 10% (v/v) pyridine (pH 9)
to terminate the reaction. The solution was
then diluted four-fold with water and 1 mL of
the diluted solution was put onto a Whatman
K5F TLC plate (20 cm width×10 cm length).
The TLC plate was then irrigated four times
with 85:15 (v/v) MeCN–water using a path-
length of 8.5 cm. Carbohydrates on the TLC
plate were detected by dipping the plate into
MeOH containing 0.3% (w/v) N-(1-naph
D-fructose,
D-glucose, sucrose, acceptor prod-
ucts, and dextran were quantitatively deter-
mined on the TLC plate by using a
PhosphoImager (Molecular Dynamics, Sunny-
vale, CA).
Assay of the
from sucrose and from dextran to maltose.
-Glucosyl transfer reaction from sucrose to
D
-glucosyl transfer-reaction
D
maltose was performed at concentrations of
10, 20, 40, and 100 mM sucrose and 20, 40,
and 80 mM maltose in 10 mM NaOAc buffer
(pH 5.2), containing 1 mg mL−1 Tween 80
Table 1
Characteristics and kinetic constants for the dextrans used to study glucosyl transfer to 50 mM maltose (forming panose) and the
hydrolysis (forming -glucose)
D
Dextran characteristics
Dextrans T-10
T-40
T-500
a
Mw a
9,700
6,000
5%
0.17
0.48
42,500
26,500
5%
0.038
0.35
465,000
191,500
5%
0.005
0.30
Mn
Degree of branching (DB) a,b
Reducing ends c
Non-reducing ends c
Kinetic constants for the glucosyl transfer from dextran chain to maltose
Dextrans T-10
T-40
0.5390.11
1.9090.10
T-500
0.9690.12
2.0090.10
Kmapp (g L−1
kcatapp (s−1
)
0.9790.16
2.4090.10
)
Kinetic constants for hydrolysis of glucosyl residues from the non-reducing ends of dextran chains
Dextrans T-10
4792
T-40
2791
0.09090.002
T-500
1992
0.08590.002
Km (mg mL−1
Kcat (s−1
)
)
0.08690.002
a The number-average molecular weight, the weight-average molecular weight (Mn and Mw) and the degree of branching (DB)
were obtained from the technical bulletin provided by Pharmacia.
b Degree of branching is defined as (branching glucose unit)/(total glucose unit)×100.
c The numbers of reducing ends and non-reducing ends are given in mmol g−1 of dextran and were calculated using the
following relationships: reducing ends=1000/Mn; non-reducing ends=[(1000Mn/162)(DB/100)+1000]/Mn.

186
M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
thyl)ethylendiamine and 5% (v/v) H2SO4, fol-
lowed by heating the plate at 120 °C for 20
min [10]. The increase in the acceptor product,
panose, in the early stages of the reaction was
measured on the TLC plate by using a densit-
ometer (model GS-670, BioRad, Hercules,
CA) from which the initial rate of the acceptor
reaction was determined [11].
Assay of dextran hydrolysis. Hydrolysis of
dextran by dextransucrase was studied at four
concentrations of dextran (0.05, 0.1, 0.2, and
0.5 mg mL−1) using three kinds of dextran
(T-10, T-40 and T-500) in 10 mM NaOAc
buffer (pH 5.2), containing 1 mg mL−1 Tween
80 and 1 mM CaCl2 with 3.1 IU mL−1 dex-
transucrase at 21 °C. Aliquots (600 mL) were
taken every 30 min for a total of 120 min and
the reaction was stopped by the addition of
140 mL of 0.2 M NaOH containing 0.1 M
Tris–HCl. After 10 min, the digest was neu-
tralized by adding 110 mL of 0.2 M HCl. A
200 mL-portion of the neutralized solution was
then put into the well of a microtiter plate and
the amount of glucose determined using glu-
cose oxidase [12].
Fig. 1. Lineweaver–Burk plot of the formation of dextran in
the absence and presence of maltose. Symbols ( , ꢀ, ꢁ)
represent the reaction with 0, 20, and 40 mM maltose, respec-
tively.
tose at sucrose and maltose concentrations of
12.5 mM each; Fig. 2(B) is the time course for
sucrose and maltose concentrations of 50 mM
each. The decrease in the amount of dextran
with the concomitant increase in the amount
of acceptor products is apparent in the first
200 min of the reaction, especially for the low
concentrations of the substrates. There was
also an increase in the DP of the acceptor
products in the early stages (200 min), fol-
lowed by a decrease in the DP in the later
stage (after 500 min). This was especially ap-
parent in the reaction with the higher amount
(42 IU mL−1) of enzyme with both the 12.5
mM (Fig. 2(A)) and the 50 mM (Fig. 2(B))
concentrations of the substrates. The increase
in the amount of acceptor products with the
decrease in the amount of dextran in the early
stages of the reaction suggests the possibility
Determination of kinetic constants. The ki-
netic
equations
were
the
standard
Lineweaver–Burk equations for one and two
substrate reactions and for competitive, non-
competitive, and mixed inhibition reactions
[13]. Kinetic constants (Km and kcat) were ob-
tained by plotting the data 1/6i versus 1/[S]
and by using a curve-fitting method of least-
squares regression analysis [14].
3. Results
Maltose inhibition of dextran synthesis.—
The Lineweaver–Burk plots of 1/6i versus 1/
[Suc] for the formation of dextran in the
presence and absence of maltose are shown in
Fig. 1. From the types of lines obtained, the
inhibition pattern was determined to be
mixed-type inhibition. The kinetic constants
that were determined are given in Table 2. The
KSmuc was 13.2 mM, very close to that previ-
ously reported (9 mM) [15].
that
D
-glucose residues of dextran were being
transferred to the acceptor. The decrease in
both dextran and the acceptor in the later
stages, especially for the 12.5 mM concentra-
tion of substrates and high concentration of
the enzyme (Fig. 2(A)) suggests the possibility
that both dextran and the acceptor products
were being hydrolyzed.
Time course of the maltose acceptor reac-
tion.—Fig. 2(A) illustrates the time course of
the transfer of -glucose from sucrose to mal-
D

M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
187
Dex
cat
Kinetic analysis of the transfer of
D
-glucose
k
) for the three molecular sizes of dextran
with 50 mM maltose are given in Table 1. The
data show that the kinetic constants were not
significantly affected by differences in the
molecular weights of the dextrans.
from sucrose to maltose.—Panose was the
only acceptor product formed in the early
(initial velocity) stages of the transfer reaction
of
D
-glucose from sucrose to maltose. The
The mechanism of the reaction of dextran
with maltose was further studied, using B-
512F-dextran T-500. The Lineweaver–Burk
plot, 1/6i versus 1/[dex], for the reaction is
shown in Fig. 4. The parallel lines of this plot
are consistent with a ping-pong/bi-bi mecha-
nism. The kinetic parameters are given in
Table 2. The KMm al (29.2 mM) is essentially
identical to the KMm al (31.4 mM) obtained for
Lineweaver–Burk plot, 1/6i versus 1/[Suc], for
the formation of panose is shown in Fig. 3.
The parallel lines obtained for this plot are
consistent for a ping-pong/bi-bi mechanism.
The kinetic parameters for this reaction are
given in Table 2. The kcat value of the acceptor
reaction is 1.67 times larger than that of the
dextran-forming reaction. The KSmuc value
(13.2 mM) is nearly identical to that observed
for the formation of dextran (9 mM).
the transfer of -glucose from sucrose to mal-
D
tose. The kcat, however, is very low, 0.5% of
the kcat for the formation of dextran from
sucrose and 0.3% of the kcat for the formation
Kinetic analysis of the transfer of
D
-glucose
from dextran to maltose.—In this experiment,
only maltose and dextran were incubated to-
gether with dextransucrase. Panose was the
only product observed in the early stages of
the reaction. The kinetic constants (KDmex and
of panose from the transfer of -glucose from
D
sucrose to maltose. These results suggest that
because maltose has the same Km for the two
reactions, maltose is binding at the same bind-
ing site (the acceptor binding site) in the two
reactions. The low kcat for the transfer of
Table 2
Kinetic constants involved in the maltose acceptor reactions
and dextran inhibition and hydrolysis reactions
D-glucose from the dextran chain to maltose
indicates that the formation of the glucosyl–
enzyme covalent complex by the transfer of
Synthesis of dextran with maltose as an inhibitor a
D
-glucose from dextran to the enzyme is a
KSmuc
Ki,s
Ki,i
13.290.8 mM
21.192.2 mM
65.398.6 mM
641914 s−1
poor reaction in contrast to the transfer of
D-glucose from sucrose to maltose.
kcat
Dextransucrase hydrolysis of dextran.— -
D
Glucose is the only hydrolysis product in the
early stage of the reaction. Kinetic parameters
Glucosyl transfer from sucrose to maltose (formation of
panose) b
KSmuc
KMm al
kcat
13.192.0 mM
31.494.5 mM
1070962 s−1
of the hydrolysis of the three dextrans are
Dex
summarized in Table 1. Neither KDmex nor k
cat
was significantly affected by the molecular
Dex
cat
Glucosyl transfer from dextran T-500 to maltose
(formation of panose) c
weights of the dextrans. The k
values of the
hydrolysis reaction are very low, between
KDmex
KMm al
kcat
1.5790.21 mg mL−1
Suc
cat
0.013 and 0.014% of that of the k
for the
29.295.4 mM
synthesis of dextran. The KDmex value of dex-
tran hydrolysis reaction is significantly lower
than the KSmuc obtained in the transfer reaction
3.290.2 s−1
Hydrolysis of dextran T-500 (formation of glucose)
Km
kcat
0.01990.002 mg mL−1
0.08590.002 s−1
of
D-glucose from sucrose to maltose.
a The kinetic parameters for mixed inhibition appear in the
following equation: d[Dex]/dt=kcat [E][Suc]/{KSmuc(1+[Mal]/
Ki,s)+[Suc](1+[Mal]/Ki,i)}.
4. Discussion
b The kinetic parameters in ping-pong/bi-bi mechanism ap-
pear in the following equation: d[Pan]/dt=kcat[Suc][Mal]/
([Suc][Mal]+KSmuc[Mal]+KmMal[Suc]).
The Lineweaver–Burk plot for the synthesis
of dextran from sucrose, in the presence of
maltose, indicates that maltose is a mixed-type
inhibitor (Fig. 1), which is essentially a non-
c The kinetic parameters in ping-pong/bi-bi mechanism ap-
pear in the following equation: d[Pan]/dt=kcat[Dex][Mal]/
([Dex][Mal]+KDmex[Mal]+KmMal[Dex]).

188
M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
Fig. 2. Time course of the maltose acceptor reaction with (A) 12.5 mM sucrose and 12.5 mM maltose and with (B) 50 mM sucrose
and 50 mM maltose; , ꢀ, ꢁ represent the reaction with 0.42, 4.2, and 42 IU mL−1 dextransucrase, respectively. The vertical
axes of the top and the middle graphs represent the percentage of radioactivity incorporated into dextran and acceptor products,
respectively. The vertical axis of the bottom graphs represents the average degree of polymerization of the acceptor products.
tor reaction from sucrose to maltose is 1.69
times larger than the kcat value for the synthe-
sis of dextran as shown in Table 2. This
competitive inhibitor. This indicates that mal-
tose (I) binds in the acceptor binding-site [16],
forming an ESI complex in which the dissocia-
tion constant of sucrose (S) from ESI is differ-
ent from the dissociation constant of sucrose
from ES [15]. In this special kind of non-com-
petitive inhibition, both Km and kcat are al-
tered [17]. Maltose inhibits the synthesis of
supports the observation that the rate of
D
-
fructose released from sucrose is accelerated
by the addition of some acceptors to the di-
gest, as previously reported [18,19].
Su and Robyt [5] found that an increase in
the equimolar concentration of sucrose and
maltose gave a decrease in the ratio of dextran
to acceptor product. The ratio of the initial
rate of dextran formation versus the total
activity was calculated from the data in Table
2 as the values of d[Dex]/dt divided by d[Dex]/
dextran by diverting the transfer of
away from dextran and to itself. The transfer
of -glucose from sucrose to maltose in the
D
-glucose
D
acceptor reaction gave parallel lines for the
sucrose Lineweaver–Burk plot at three con-
centrations of maltose (Fig. 3), which is con-
sistent with a ping-pong/bi-bi mechanism.
This type of kinetics is characteristic of the
formation of a covalent glucosyl–enzyme in-
termediate, which previously had been postu-
lated by Robyt and co-workers [1–3,16] in the
synthesis of dextran and in the formation of
acceptor products. The kcat value of the accep-
dt+d[Pan]/dt, and the percentage of
D-glu-
cose incorporated into dextran as a function
of the equimolar concentration of sucrose and
maltose was plotted as shown in Fig. 5. The
plot of Fig. 5 indicates that the amount of
D-glucose incorporated into dextran as a func-
tion of the concentration of sucrose and mal-

M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
189
Fig. 3. Lineweaver–Burk plot of the dextransucrase-catalyzed
formation of panose from sucrose and maltose; , ꢀ, ꢁ,
represent the reaction with 20, 40, and 80 mM maltose,
respectively.
Fig. 5. Percentage of
D
-glucose incorporated into dextran as a
function of the equimolar concentrations of sucrose and
maltose. The curve was obtained using the kinetic constants
shown in Table 2.
tose decreases exponentially and explains the
substrate concentration effect on the dextran
to acceptor products ratio as observed by Su
and Robyt [5].
Su and Robyt [5] also reported that an
increase in the amounts of dextransucrase pro-
duced a decrease in the amount of dextran
and a concomitant increase in the amount of
acceptor products when the 24 h, equilibrium
amounts of products were measured. In the
present report, the kinetics of the initial stages
of these reactions, with various concentrations
of dextransucrase, were studied instead of the
late stage where equilibrium had occurred.
The early stages (200 min) of the maltose
acceptor reaction show that dextran decreased
and the acceptor products increased after su-
crose had disappeared (Fig. 2(A and B)). A
possible explanation for these observations is
that
D-glucosyl units were being transferred
from dextran to the acceptors. Such a transfer
reaction had been previously reported by
Binder and co-workers [20]. The decrease of
the dextran and the concomitant increase in
the amount of acceptor products could thus
be due to the slow transfer of
D
-glucose from
dextran to the acceptor(s). This reaction
would especially be observed when the
amount of enzyme is high but would not be as
readily observed when the amount of enzyme
is low, due to the relatively slow rate of
transfer.
The study of the transfer of
D
-glucose from
Fig. 4. Lineweaver–Burk plot of the dextransucrase-catalyzed
formation of panose from dextran T-500 and maltose;
dextran to maltose was performed by using
B-512F dextran T-500. It was found that the
reaction was much slower than both the syn-
ꢁ, ꢀ,
represent the reaction with 20, 40, and 80 mM
maltose, respectively.

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M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
thesis of dextran from sucrose and the transfer
reaction of -glucose from sucrose to maltose.
cosidic bonds between
D
-glucose residues,
D
with a kcat of 641 s−1. The second reaction
forms a-(1“3) branch linkages by the trans-
fer of glucosyl or dextranyl units from the
active site to an exogenous dextran chain [3].
The third reaction, which occurs in the pres-
ence of sucrose and another carbohydrate, an
acceptor (in this study, maltose), gives the
Transfer, however, did occur and panose was
formed. The kinetics gave parallel lines (Fig.
4), indicating that the mechanism is also ping-
pong/bi-bi and suggests the formation of the
same covalent glucosyl–enzyme intermediate
that was formed in both the maltose acceptor
reaction and in the synthesis of dextran. Fur-
ther, by using three B-512F dextrans of differ-
ent molecular weights, no significant
differences were observed in the kinetic con-
stants of the transfer reaction (Table 1). This
shows that the transfer was occurring from
the non-reducing ends of the dextran chain
because the number of non-reducing ends is
not significantly affected by its molecular
weight. If the transfer had been from the
reducing end of dextran, the rates of reaction
would have been inversely proportional to the
molecular weights of the dextrans. The en-
transfer of
D
-glucose to maltose with a kcat of
1070 s−1. The fourth reaction, which occurs
between dextran and maltose in the absence of
sucrose, gives the transfer of
the non-reducing ends of the dextran chains to
maltose with a relatively low kcat of 3.2 s−1
D
-glucose from
.
The fifth reaction, which is exclusively with
dextran, gives the extremely slow hydrolysis of
D
-glucose from the non-reducing ends of the
dextran chains, with a very low kcat of 0.085
s−1
.
The ping-pong/bi-bi kinetics that were ob-
served for the reactions suggest that all of the
reactions take place through the formation of
a covalent glucosyl–enzyme intermediate.
When the amount of enzyme is relatively high,
the products from the fourth and fifth reac-
tions are observed. Further, as the concentra-
tions of the substrates in the third reaction (in
this study, sucrose and maltose) are increased,
there is an exponential decrease in the amount
of dextran formed (Fig. 5). As previously re-
ported [5], as the concentration ratio of accep-
tor to sucrose changes, there is a change in the
amount of dextran formed and the amount of
acceptor product formed. At low maltose to
sucrose ratios, dextran and a homologous se-
ries of acceptor products are formed. As the
acceptor to sucrose ratios are increased, there
is a decrease in the amount of dextran formed
and an increase in the amount of acceptor
product formed but a decrease in the number
of acceptor products in the homologous series.
At some ratio, no dextran is formed and only
a single acceptor product is formed.
zyme, thus, transfers -glucosyl units from the
D
non-reducing ends of dextran to maltose via a
covalent glucosyl–enzyme intermediate.
The enzyme also catalyzes the hydrolysis of
dextran, but at an extremely low rate. Because
the Km values for the hydrolysis did not sig-
nificantly vary with the molecular weights of
the dextrans, it is concluded that the enzyme
hydrolyzes
D
-glucosyl residues from the non-
reducing ends of dextran chains. The Km value
of dextran T-500 (meaning the apparent
Dex
mapp
Michaelis constant for dextran [K
] at
[H2O]=55 M) was 0.019 mg mL−1, a much
smaller value than that obtained in the trans-
fer reaction of -glucose from dextran to mal-
D
tose. From these results, the theoretical Km
value for H2O (KHm O) in the hydrolysis is
2
calculated to be 4490 M using the following
ping-pong/bi-bi mechanism equation:
KDmeaxpp =KDmex [H2O]/(KmH O+[H2O])
2
When KmDaexpp =0.019 mg mL−1, KmDex=1.57
mg mL−1 (Table 2), and [H2O]=55 M, thus,
showing that the enzyme has a very low
affinity for water at its active site and there-
fore will catalyzes hydrolysis extremely slowly.
The present results explain the previously
unexplained observations [5] that the equi-
librium amount of dextran formed to the equi-
librium amount of acceptor product(s) formed
decreases as the concentrations of sucrose and
maltose increase and as the concentration of
enzyme increases in the digest. The effect of
the sucrose and maltose concentrations is due
In
summary,
L.
mesenteroides
B-
512F(MC16) dextransucrase catalyzes five dif-
ferent reactions. The first reaction synthesizes
dextran from sucrose and forms a-(1“6) gly-

M. Kitaoka, J.F. Robyt / Carbohydrate Research 320 (1999) 183–191
191
279–286.
to the more favorable kcat for the acceptor
reaction for maltose over the kcat for the syn-
thesis of dextran. The effects of increasing the
amount of enzyme are not due to an impossi-
ble condition of a shift in the equilibrium of
the reaction by the catalyst, but to the transfer
[5] D. Su, J.F. Robyt, Carbohydr. Res., 248 (1993) 339–348.
[6] M. Kitaoka, J.F. Robyt, Enzyme Microb. Technol., 22
(1998) 527–531.
[7] M. Kitaoka, J.F. Robyt, Enzyme Microb. Technol., 23
(1998) 386–391.
[8] A.W. Miller, J.F. Robyt, Biochim. Biophys. Acta, 785
(1986) 89–96.
[9] A.W. Miller, J.F. Robyt, Biochim. Biophys. Acta, 880
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[10] R. Mukerjea, D. Kim, J.F. Robyt, Carbohydr. Res., 292
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[13] D.L. Purich, in D.L. Purich (Ed.), Contemporary Enzyme
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[14] W.W. Cleland, Methods Enzymol., 63 (1979) 103–138.
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of
D
-glucose from the non-reducing ends of
the dextran chains to maltose when the
amount of enzyme is increased to a relatively
high level, and the decrease of dextran is
further due to the hydrolysis of
D
-glucose
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dextran chains when high amounts of enzyme
are used.
[16] D. Su, J.F. Robyt, Arch. Biochem. Biophys., 308 (1994)
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