Effect of sucralose on the enzyme kinetics of invertase using real-time NMR spectroscopy and progress curve analysis

Article abstract of DOI:10.1016/j.carres.2017.10.019

Sucralose, a derivative of sucrose, is widely used in noncaloric artificial sweeteners (NAS). Contrary to the belief that sucralose is physiologically inert and a healthy alternative sweetener to natural sugar, emerging studies indicate that sucralose alters the host metabolism as well as the composition of the microbiome. In this manuscript, we use real-time nuclear magnetic resonance (NMR) spectroscopy to demonstrate that sucralose alters the enzymatic conversion of sucrose to glucose and fructose. The real-time NMR progress curve analysis suggests that sucralose has the characteristic of a competitive inhibitor on the kinetics of the enzymatic process. This affects the rate of glucose production, and thus indirectly affecting the mutarotation process of α-D-glucose to β-D-glucose conversion. At a 1:2 M ratio of sucrose to sucralose, the results show that the catalytic efficiency of the enzyme is reduced by more than 50% in comparison to the measurements without sucralose. Altogether, as sucralose alters the rate of glucose production, sucralose cannot be considered inert to the metabolism as several downstream events in both prokaryotic and eukaryotic systems strongly depend on the rate of glucose metabolism.

Full text of DOI:10.1016/j.carres.2017.10.019

Carbohydrate Research 455 (2018) 5e9
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Effect of sucralose on the enzyme kinetics of invertase using real-time
NMR spectroscopy and progress curve analysis
a
a
a, b, *
Cheenou Her , Jaideep Singh , V.V. Krishnan
a
Department of Chemistry, California State University, Fresno CA 93740, United States
Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Davis CA 95616, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2017
Received in revised form
Sucralose, a derivative of sucrose, is widely used in noncaloric artificial sweeteners (NAS). Contrary to the
belief that sucralose is physiologically inert and a healthy alternative sweetener to natural sugar,
emerging studies indicate that sucralose alters the host metabolism as well as the composition of the
microbiome. In this manuscript, we use real-time nuclear magnetic resonance (NMR) spectroscopy to
demonstrate that sucralose alters the enzymatic conversion of sucrose to glucose and fructose. The real-
time NMR progress curve analysis suggests that sucralose has the characteristic of a competitive inhibitor
on the kinetics of the enzymatic process. This affects the rate of glucose production, and thus indirectly
19 October 2017
Accepted 27 October 2017
Available online 31 October 2017
Keywords:
affecting the mutarotation process of a-D-glucose to b-D-glucose conversion. At a 1:2 M ratio of sucrose
Enzyme kinetics
NMR spectroscopy
Progress curve analysis
Sucralose
to sucralose, the results show that the catalytic efficiency of the enzyme is reduced by more than 50% in
comparison to the measurements without sucralose. Altogether, as sucralose alters the rate of glucose
production, sucralose cannot be considered inert to the metabolism as several downstream events in
both prokaryotic and eukaryotic systems strongly depend on the rate of glucose metabolism.
Sucrose
©
2017 Elsevier Ltd. All rights reserved.
1
. Introduction
investigated the effect of sucralose intake in humans and organ-
isms. Sucralose has been shown to alter glycemic and insulin re-
sponses, which are parts of the glucose metabolic process following
glucose ingestion [7]. A study done by Chia et al. showed that in-
dividuals using sucralose, and other NAS, resulted in weight gain/
obesity, which is the opposite of what users are expecting [8].
Omran et al. showed that the environmental microbial population
is incapable of breaking down sucralose, and further suggested that
sucralose has the characteristics of a competitive inhibitor during
the hydrolysis of sucrose via invertase [9]. Other recent studies
have indicated that sucralose activates an ERK1/2-ribosomal pro-
Non-caloric artificial sweeteners (NAS) are used as sugar sub-
stitutes that provide a sweet taste without added calories or gly-
cemic effects in dietary products. Popular NAS use the sucrose
derivative 1, 6-Dichloro-1, 6-dideoxy-b-D-fructofuranosyl-4-chloro-
4-deoxy-a-D-galactopyranoside, commonly known as sucralose, a
disaccharide that has a similar structure to sucrose [1,2]. The dif-
ference between the structures are three of the hydroxyl groups, at
position C4, C1 , and C6’ (atom labeling follows the recommenda-
0
tions of the IUPAC-IUB Commission on Biochemical Nomenclature
[
3]), are replaced with chlorine atoms in sucralose. Sucralose is
tein S6 signaling axis in MIN6 cells (pancreatic b cell lines for
ꢀ
thermally stable up to 119 C in its pure form, is stable throughout a
wide pH range (~pH 3e7), approximately 650 times sweeter than
sucrose, and does not decompose in long term storage [1,4e6].
These characteristics make sucralose attractive to the food market
as an artificial sweetener additive.
glucose metabolism and glucose-stimulated insulin secretion) [10],
increases antimicrobial resistance [11], and suppresses food intake
by inducing a fasting response in fruit flies [12,13].
The enzymatic conversion of sucrose to glucose and fructose via
invertase is one of the most important enzymatic reactions in
prokaryotic and eukaryotic cells. This enzyme catalyzed reaction
follows the classic Michaelis-Menten model, a well-studied system
Due to these attractive natures of sucralose, many studies have
[14]. Recently, we have implemented an experimental method to
monitor the hydrolysis of sucrose via invertase using nuclear
magnetic resonance (NMR) spectroscopy, since the chemical shift
of the substrate (sucrose) is well defined from the product (glucose)
*
Corresponding author. Department of Chemistry, California State University,
Fresno CA 93740, United States
E-mail
addresses:
krish@csufresno.edu,
vvkrishnan@ucdavis.edu
(
V.V. Krishnan).
https://doi.org/10.1016/j.carres.2017.10.019
008-6215/© 2017 Elsevier Ltd. All rights reserved.
0

6
C. Her et al. / Carbohydrate Research 455 (2018) 5e9
[
(
15,16]. This approach allows the conversion of sucrose to glucose
-D-glucose) and fructose via invertase to be monitored in real-
d½Pꢂ
d½Sꢂ
V
max½Sꢂ
a
n
¼
¼ ꢃ
¼ K
[1]
dt
dt
þ ½Sꢂ
time, and the subsequent mutarotation process that converts
D-glucose to -D-glucose. In this manuscript, we extend this pro-
a
-
M
b
Where [S] is the substrate concentration, Vmax is the maximal
rate of enzymatic turnover (sucrose to glucose and fructose), and
represents the Michaelis-Menten half-saturation constant. The
first-order differential equation (Equation [1]) can be integrated to
obtain the integral form of the MichaeliseMenten equation
cedure to demonstrate the role of sucralose in altering the
Michaelis-Menten kinetics of sucrose hydrolysis via invertase. The
combination of real-time NMR measurements and progress curve
analysis is used to determine the Michaelis-Menten constant (K
and maximum velocity (Vmax). The results from the progress curve
analysis indicate that sucralose has the characteristic of
K
M
M
)
[
20e22] as shown in Equation [2]:
a
ꢀ½
ꢁ
competitive inhibitor to invertase, which significantly reduces the
catalytic efficiency of the enzyme.
Sꢂ
0
KM ln
þ ½Sꢂ ꢃ ½Sꢂ ¼ Vmax
t
[2]
0
½
Sꢂ
The Lambert-W function is a mathematical function, in the form
of an exponential function, and has several applications in com-
puter science, mathematics, and physical sciences [23,24]. Mathe-
matically, the exponential function and the natural logarithmic
function, ln(x), are exponentially related. Similarly, W(x) is defined
2
. Materials and method
Invertase (EC 3.2.1.26,
purchased from Sigma-Aldrich with a specific activity of >300 u/mg
of solid (pH 4.6, 303 K). Sucrose, D-(þ)-Glucose, D O (99.9 atom %
D) and 3-(Trimethylsilyl) propionic-2, 2, 3, 3-d acid sodium salt
TSP) were also purchased from Sigma-Aldrich. Sucralose was
purchased from Alfa Aesar.
b-fructofuranosidase, S. cerevisiae) was
y
2
as the inverse of the function satisfying ye ¼ x and its solution
4
expressed by the Lambert-W(x) function like y ¼ W(x).
(
By substituting y ¼ ½Sꢂ=K in Equation [2] and rearranging, we
M
get Equation [3]:
ꢀ½
ꢀ
ꢁꢁ
Sꢂ ꢃ Vmax
t
½Sꢂ
y
0
0
ye ¼ xðtÞ ¼ exp
þ ln
tꢁ
2
.1. Sample preparation
K
M
K
M
ꢀ
[3]
½
Sꢂ
½Sꢂ ꢃ Vmax
0
0
For all the NMR kinetics experiments, pH 5.0 was used. The
sucrose (80 mM) and invertase (5 g/mL) concentrations were kept
¼
exp
K
K
M
m
M
constant, while the sucralose concentrations varied. The sucralose
concentrations used were 2 mM, 10 mM, 20 mM, 40 mM, 80 mM,
The left-hand side of Equation [3] is analogous to the Lambert-
W function in the Corless et al., 1996 article [24]. Thus, using the
definition of the Lambert-W function (y ¼ W(x)), an expression for
y can be obtained as that expressed in Equation [4]:
120 mM, and 160 mM. A timer was set at the beginning of the
addition of the invertase solution to account for the delay time (the
time before the collection of the NMR spectra).
ꢂ½
ꢃ
Sꢂ
À
Á
0
y ¼ W
exp ½Sꢂ ꢃ Vmaxt=KM
[4]
0
K
M
2.2. Real-time NMR measurements of the hydrolysis of sucrose via
invertase
Further substituting y ¼ ½Sꢂ=K
M
back in Equation [4], we get
Equation [5]:
A Varian-Agilent 400 MHz NMR spectrometer was used to
collect the NMR data. Each 1D-proton NMR spectrum was collected
with a spectral width of 14.88 ppm over 32768 number of points
ꢂ½
ꢀ
tꢁꢃ
Sꢂ
½Sꢂ ꢃ Vmax
o
o
½
Sꢂ ¼ K W
exp
[5]
M
K
K
M
M
(
with Ernst angle [17]). Each spectrum was signal averages over 24
Equation [5] relates the substrate concentration at any time ([S])
to its initial concentration ([S] ), the MichaeliseMenten kinetic
parameters Vmax and K . Equation [5] is used to fit the real-time
experimental data obtained for enzyme kinetics using the anal-
ysis code written in the R-Statistical environment [25].
transients and 1s relaxation delay between the transients leading
to a total time of 90.0 s per experiment (24 ꢁ 3.75 s per transient).
The 1D-proton NMR spectra were collected in an array fashion one
after another and were collected as necessary to follow the reaction
to completion. The NMR data were processed using the program
MNova NMR™. The area under the curve of the sucrose resonance
at 5.41 ppm), alpha-D-glucose and beta-D-glucose resonance (at
.22 ppm and 4.64 ppm), and sucralose resonance (at 5.49 ppm)
were used to calculate their concentration using TSP (at 0 ppm) as
the standard. Progress curve analysis was done following the pro-
cedure in the following reference [16].
0
M
3
. Results
(
5
3
.1. NMR spectral differentiation between sucrose, glucose, and
sucralose
Glucose, sucrose, and sucralose all have at least one proton with
a distinct resonance, granting the ability to perform real-time
analysis of the enzyme kinetics via NMR spectroscopy. Fig. 1
shows the NMR spectra of sucrose (Fig. 1a), sucralose (Fig. 1b)
and glucose (Fig.1c). The C1 proton for sucrose shows a double peak
(doublet) close to the resonance frequency of 5.41 ppm, while the
same proton in the sucralose is shifted downfield to 5.49 ppm. Also,
the C5 and C4 protons of sucralose resonate at 4.42 ppm and
4.54 ppm, respectively (Fig. 1b). As shown in Fig. 1c, the NMR
spectrum for glucose depicts a doublet at 5.22 ppm (C1 proton) [26]
2
.3. Progress curve analysis of the real-time NMR data
The catalyzed breakdown, or hydrolysis, of sucrose, can be
shown by the Michaelis-Menten equation. Schnell and Mendoza
22] presented the integrated form of the Michaelis-Menten
[
equation using the Lambert-W function with an application
developed by Goudar and co-workers [18,19]. A brief description of
enzyme kinetics is given here following our earlier work [16]:
The MichaeliseMenten equation in the differential form can be
used to describe the dynamics of substrate depletion as Equation
for
-D-glucose. The relative intensities of the
glucose peaks (Fig. 1c) are dependent on the thermodynamic
a-D-glucose and the second doublet at 4.64 ppm (C1 proton) for
b
a
-D-glucose and -D-
b
[1]:

C. Her et al. / Carbohydrate Research 455 (2018) 5e9
7
Glucose
(
c)
(b)
Sucralose
Sucrose
(a)
5.5
5.0
4.5
1H ppm
Fig. 1. Differentiation of sucrose, glucose, and sucralose nuclear magnetic resonance
NMR) spectra in the absence of enzymatic activity. (a) Expanded region of the one-
(
dimensional NMR spectrum of sucrose the substrate. (b) Expanded region of the
NMR spectrum of sucralose, where the peak has shifted downfield due to change in the
chemical structure. (c) Expanded region of the NMR spectrum of glucose showing both
the
a- (downfield) and b- (up-field) anomers at their ambient temperature equilibrium
populations.
Fig. 2. Real-time qNMR results of substrate depletion ([S]) and product formation
(
[P]
symbols) is shown as a function of time due to the hydrolysis process of the enzyme
invertase. Anomerization of to the anomer of glucose is also observed, depicting the
a
). The concentration of sucrose (panel a, black symbols) and glucose (panel b, black
equilibria of the glucose anomers in solution. At ambient temper-
ature, the equilibrium favors the existence of the more stable -D-
a
b
b
dynamic equilibrium of the mutarotation process. In the presence of sucralose, both
the substrate (red symbols, panel a) and product (red symbols, panel b) are altered. The
concentration of sucralose does not change during the reaction (blue symbols) but
alters the rate of hydrolysis of sucrose via invertase. (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this
article.)
glucose anomer (~64%), as depicted by the higher peak intensity in
Fig. 1c [27]. The distinct, well-resolved peaks of sucrose, sucralose,
and glucose enable the differentiation between molecules in the
same sample, allowing for the determination of kinetic rates of
conversion by the enzyme catalysis.
3
.3. Effect of sucralose of the enzymatic rate of conversion of
3.2. Real-time enzymatic conversion of sucrose to glucose
sucrose
With the known concentration of the internal reference (TSP),
Though the sucralose concentration remains constant during
the integration of the area under the respective peaks of sucrose
[S]), glucose ([P]) and sucralose allowed for the determination of
the rate of change of the reactant and product concentrations
Fig. 2). Fig. 2a illustrates the plot of the real-time hydrolysis of the
sucrose ([S]) via invertase, without sucralose (filled black circle)
and in the presence of sucralose (filled red squares). While the
hydrolysis of sucrose occurred in both conditions, the concentra-
tion of sucralose (blue diamonds) remained constant in the pres-
ence of invertase.
the experiment (blue diamond symbols Fig. 2a), the presence of
sucralose significantly alters the rate at which sucrose is enzy-
matically hydrolyzed via invertase. The enzymatic conversion of
sucrose to glucose and fructose follows the standard Michaelis-
Menten enzyme kinetics mechanism [28e30]. The enzymatic re-
action can be monitored either regarding the substrate (sucrose) or
terms of the product (glucose) concentration. The characteristic of
the enzyme action, in the absence of any inhibitory process, is
measured by the Michaelis-Menten parameters defined by the
(
(
Upon creation of
tion with water leads to the mutarotation of
glucose ([P] ), as shown in Fig. 2b, until an equilibrium is reached
between - and -anomers of glucose. This thermodynamic equi-
librium between both glucose anomers is temperature-dependent
and is the reason the concentration profile of -D-glucose de-
creases as the -D-glucose anomer is formed at room temperature
Fig. 2b) [27]. Mutarotation of anomers are inherent to carbohy-
drates, and most of the carbohydrates exist in multiple anomeric
forms (predominantly in and -anomers in the case of glucose).
a
-D-glucose ([P]
a
) from sucrose, the interac-
M
Michaelis-Menten constant (K ) and maximal velocity (Vmax).
a
-D-glucose to -D-
b
Using the Lambert-W function, described in equation [5], to fit
the sucrose progress curve on time, the Michaelis-Menten constant
and maximum velocity can be determined directly from a single
NMR experiment [16]. For pure enzyme kinetics (no sucralose), the
b
a
b
a
direct method yields a K
M
of 69.21 mM ± 1.24 mM and Vmax of
b
3
.52 ± 0.04 mM/min. As shown in Fig. 2a, sucralose slows down the
(
conversion of sucrose to
the direct method yields a K
a
-D-glucose. In the presence of sucralose,
of 116.81 mM ± 2.09 mM and Vmax of
M
a
b
and 3.78 mM/min ±0.05 mM/min. Progress curve analysis produces
fairly consistent results with an average coefficient variation (co-
However, the co-existence of these multiple anomeric forms is due
to solvation effects and is independent of the enzyme action.
The current experimental parameters are chosen such that there
is enough time-resolution particularly at the early time points to
efficiently determine the enzyme kinetics and the S/N required
analyzing the data. Furthermore, the kinetic measurements focus
more on the rates (decay of sucralose or growth of glucose) rather
than the absolute concentration; we assume that shorter relaxation
delay (3.75 s per transient) with the Ernst angle would not bias the
results presented here.
efficient variation (CV) in %) of K
M
measured from the three trials is
9
%, and that of the Vmax is 7% [16].
3.4. The effect of sucralose at varying concentration on the
enzymatic conversion of sucrose
The presence of sucralose, even at lower concentrations, affects
the catalytic mechanism of invertase. Fig.
3 shows the

8
C. Her et al. / Carbohydrate Research 455 (2018) 5e9
Fig. 4. Sucralose concentration effect on enzymatic activity. Variation of catalytic ef-
ficiency (CE) as a function of the molar ratio of sucralose to sucrose concentration.
With increasing concentration of sucralose, the effectiveness of the enzymatic reaction
drops significantly. The continuous red line shows the exponential regression and the
dotted blue lines shows the 95% confidence interval of the fit. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
efficiency of the enzyme function is significantly decreased, more
or less in an exponential fashion with increasing concentration of
sucralose. An exponential fit (Fig. 4), suggests that the catalytic
ꢃ1
ꢃ1
efficiency (in units of mM min ) reduces at a rate of decreases by
5
0
0% for every increase in the molar ratio of sucralose to sucrose by
.87 ± 0.15.
Fig. 3. The hydrolysis of sucrose via invertase in the presence of varying sucralose
concentration. (a) The rate of hydrolysis of sucrose and (b) rate of formation of a-D-
glucose as a function of varying the concentration of sucralose. The sucralose con-
centrations are noted in the legends.
4. Discussion
The first step in the metabolism of sucrose is the conversion to
simpler, soluble monosaccharide forms (glucose and fructose).
Glucose (or fructose) can be transported across the intestinal wall
and delivered to the tissues, but this transportation process de-
pends primarily on the production rate of glucose (or fructose) by
enzyme kinetics. The rate of metabolic flow (also known as flux)
through the homeostatic mechanisms pathway is high, but the
concentration of substrate/intermediates/products remains con-
stant. If this steady state is disrupted by an external change on the
rate of glucose formation, the pathway will change, and regulatory
mechanisms will be triggered. This will cause the organism to try
arriving at a new steady state to achieve homeostasis, the complex
dynamics between the glucose and other elements in the cell that
maintain a relatively constant blood sugar level [32]. Therefore,
from a simple coupled reaction point of view, the rate of glucose
production is expected to affect the energy homeostasis and the
rest of the downstream pathways related to glucose metabolism.
In the presence of sucralose, the enzyme kinetics of invertase
and sucrose was altered, thus affecting the production rate of
glucose. This effect on the early stages of sugar metabolism could
result in a cascade of downstream effects since the presence of
glucose in the blood triggers the release of insulin. Insulin is
important in regulating many other pathways that allow for the
distribution of glucose to muscle and fat cells via glucose trans-
porters. The level of insulin, which is triggered by the presence of
glucose in the blood, also plays a major role in determining
whether the glucose could be stored as glycogen, an energy storage
in the cell. When the cells need additional energy (glucose) the
hormone glucagon is released to signal the breakdown of glycogen
to glucose in the blood.
concentration-dependent effect of sucralose on invertase hydro-
lyzing sucrose into
shown in Fig. 3 the concentrations of sucralose studied were 0 mM
no sucralose), 2 mM, 10 mM 20 mM, 40 mM, 80 mM, 120 mM, and
60 mM. Table 1 shows the enzyme's kinetic parameters estimated,
a
a-D-glucose ([P] ) and fructose (not shown). As
(
1
from the progress curve analysis. The higher the concentration of
sucralose present during the reaction, the slower the hydrolysis
rate of sucrose. The presence of sucralose also had an indirect
consequence. Since the rate of formation of
a-D-glucose was
altered, the mutarotation rate of
also affected (data not shown).
a-D-glucose to b-D-glucose was
The real-time NMR-based progress curve analysis demonstrates
the deleterious effects of sucralose in the overall enzyme kinetics
process. Using the definition by Koshland [31], the catalytic effi-
ciency of the enzyme activity is determined (Fig. 4). Catalytic effi-
ciency provides insight into enzymatic mechanisms and the
functional effects of sucralose concentrations on activity. The roles
of many enzymes in a biological system, which relates to the
chemical kinetics and equilibrium processes, are essential for the
overall function in many critical biochemical reactions. The overall
Table 1
Effect of sucralose on the Michaelis-Menten kinetics using direct NMR method.
Sucralose (mM)
Sucrose: Sucralose
K
M
(mM)
Vmax (mM/min)
0
2
1:0
4:1
8:1
4:1
2:1
1:1
1:1.5
1:2
69.21 ± 1.24
74.61 ± 1.31
94.13 ± 2.06
87.28 ± 1.41
100.60 ± 1.71
116.81 ± 2.09
129.80 ± 2.68
155.10 ± 4.58
3.52 ± 0.04
3.62 ± 0.04
4.38 ± 0.07
3.75 ± 0.02
3.86 ± 0.05
3.78 ± 0.05
3.51 ± 0.05
3.54 ± 0.08
1
2
4
8
1
1
0
0
0
0
20
60
M
The physical meaning of K (concentration units) represents the
state when half the active sites of the enzyme are occupied by the
substrate. When the enzyme has a low affinity for the substrate, a
higher concentration of substrate molecules is required to saturate

C. Her et al. / Carbohydrate Research 455 (2018) 5e9
9
M
half the active sights leading to a high K value. On the other hand,
if the enzyme has a higher affinity to the substrate then the sub-
strate does not have to occupy a large number of active sites, a small
M M
number will suffice to lead to a low K value. The increase in the K
values with an increase in the sucralose concentration suggests
reduced affinity to the substrate, sucrose, due to the presence of
sucralose. The maximal velocity, Vmax represents how fast the
enzyme can catalyze the reaction-the number of substrate mole-
cules being catalyzed per unit time. The average value of the Vmax
was not altered significantly (3.74 ± 0.27 mM/min, Table 1), sug-
gesting that sucralose would not change the catalytic function of
the enzyme. The catalytic efficiency (Fig. 4) drops as much as 50%
for every 0.87 ± 0.15 equimolar increase of sucralose on sucrose
stability and thermal decomposition of sucralose, Ecl
1e26.
eꢀ tica Quím. 34 (2009)
2
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(
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sucralose concentration, is one indication that sucralose may be a
competitive inhibitor. The molecular structure of sucralose gives
insight into how it interacts with the active site of invertase,
facilitating competitive inhibition of sucrose hydrolysis. Sucralose
has a similar structure to sucrose, giving it the potential to bind to
the active site of invertase; however, hydrolysis does not occur. This
is believed to be due to the negative charge repulsion between the
carboxylate anion in the active site of invertase with the chlorine
atoms on sucralose [33]. The hydrogen bonding of the three hy-
droxyl groups at the C4, C1 , and C6 positions of sucrose are crucial
for the specificity and recognition of the sucrose by invertase [34].
Since the three-hydroxyl groups of sucralose are replaced with
chlorine atoms, this results in the loss of hydrogen bonding that is
crucial in the stabilization of the intermediate complex. The
competitive nature of sucralose with the active site of the enzyme,
combined with its inability to be hydrolyzed fits the model of a
competitive inhibitor.
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[
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