Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism

Article abstract of DOI:10.1039/c5ob00796h

Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC₅₀ = 4.9 mM) and Escherichia coli GS (EcGS, IC₅₀ = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC₅₀ = 2.4 mM), while its effect on EcGS increased 50-fold (IC₅₀ = 32 μM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC₅₀ = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.

Full text of DOI:10.1039/c5ob00796h

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Selective photoregulation of the activity of
glycogen synthase and glycogen phosphorylase,
two key enzymes in glycogen metabolism
Cite this: DOI: 10.1039/c5ob00796h
Mireia Díaz-Lobo,a,b,c Jaume Garcia-Amorós,d Ignacio Fita,e Dolores Velasco,d
Joan J. Guinovarta,b,c and Joan C. Ferrer*a
Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of
energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two
enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute
adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their
catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that
consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In
the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on
rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and
subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not sig-
nificantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 µM).
Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only
slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of
kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glu-
coside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen
subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of
key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen
metabolism disorders.
Received 21st April 2015,
Accepted 21st May 2015
DOI: 10.1039/c5ob00796h
www.rsc.org/obc
proteins and lipids employing azobenzenes as photoswitch-
able chromophores.5,7,10–14 Random-chemical substitution in
Introduction
Photoswitchable compounds are becoming increasingly undefined sites of biomolecules was the strategy of the past,
popular for a number of biological applications based on the whereas the novel concepts embark on the strategy of site
reversible photocontrol of the structure of biomolecules, e.g. specific incorporation and defined conformational transitions
DNA1,2 and proteins,3,4 and of various biological functions of structural elements.15–18
such as biocatalysis,5,6 ion transport,7,8 cell adhesion9 and
We are interested in the regulation of glucose and glycogen
protein folding.10,11 In these examples, the biological activity metabolism. Glycogen is a multibranched polymer of glucose
of the modified molecules is altered by a light-triggered that serves as a carbohydrate and energy reservoir in bacteria,
change of their molecular structure.12 Most investigations fungi and animals. The polysaccharide is synthesized by the
involve chemical modification of nucleotides, peptides, action of glycogen synthase (GS), which catalyzes the suc-
cessive addition of glucose units, arising from adenosine
5′-diphosphoglucose (ADPG) or uridine 5′-diphosphoglucose
(UDPG), to the non-reducing end of a growing glycogen mole-
aDepartament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Av.
Diagonal 645, E-08028 Barcelona, Spain. E-mail: jcferrer@ub.edu
bInstitute for Research in Biomedicine (IRB Barcelona), Baldiri i Reixac 10, E-08028
Barcelona, Spain
cule. Glycogen phosphorylase (GP) is a key enzyme in glycogen
degradation. Using an inorganic phosphate as a co-substrate,
GP catalyzes the release of glucose-1-phosphate from the non-
reducing end of the polysaccharide. Some metabolic disorders,
such as diabetes, or rare diseases like Lafora disease are
characterized by an abnormal accumulation of glycogen.
Therefore, compounds that could selectively modulate the
cCIBER de Diabetes y Enfermedades Metabólicas (CIBERDEM), Madrid, Spain
dGrup de Materials Orgànics, Institut de Nanociència i Nanotecnologia (IN2UB),
Departament de Química Orgànica, Universitat de Barcelona, Martí i Franquès 1,
E-08028 Barcelona, Spain
eInstitut de Biologia Molecular de Barcelona (IBMB-CSIC), Baldiri i Reixac 10,
E-08028 Barcelona, Spain
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
activity of GS or GP would be useful as potential drugs for the back transition is slow enough and one conformation of the
treatment of diseases related to glycogen metabolism. azobenzene glucoside is found to decrease the enzyme activity
Both GS and GP belong to a very large family of enzymes, significantly more than the other, this allows for the spatial
namely glycosyltransferases (GTs), which catalyze the transfer and temporal resolution of the biological activity of the
of glycosyl residues from activated donors to diverse acceptors inhibitor.
and play crucial roles in a great variety of biological processes,
We have studied the photochromic properties of azobenz-
ranging from carbon and energy metabolism and cellular and ene glucoside 1 and its potency and selectivity as an inhibitor
molecular recognition to signal transduction and cell wall for- of the enzymatic activity of three purified GT preparations that
mation. Many of these enzymes use sugar diphosphonucleo- were readily available to us: Escherichia coli GS (EcGS) and
tides as donors and they mainly differ in the nature of the rabbit muscle GPa (RMGPa) were studied as representatives of
acceptor molecule. The design and synthesis of competitive the enzymes responsible for the synthesis and degradation of
inhibitors of GTs is fraught with difficulty, since such inhibi- glycogen, respectively. Sucrose synthase 4 (SuSy 4) from pota-
tors are required to mimic a reaction transition state that effec- toes that catalyzes the conversion of UDPG and fructose into
tively involves three distinct species: the glycosyl acceptor, the sucrose and UDP was included in the study as an example of a
nucleoside diphosphate, and the glycosyl donor.19,20 There- GT that does not act on glycogen.
fore, alternative strategies to inhibition of GTs that act upon
and produce polymeric molecules have been employed. For
example, partial inhibition of the biosynthesis of cellulose19 or
Results and discussion
Synthesis of the azobenzene glucoside 1
the galactan component of the Mycobacterium tuberculosis cell
wall21 has been attained by the use of glycosyl donor analogues
that act as chain terminators. Nevertheless, it seems reason-
Synthesis of compound 1 (Fig. 1A) started with the quantitative
able that a putative competitive inhibitor aimed to act selec-
peracetylation of D-glucose with acetic anhydride in pyridine.29
tively on GS or GP should possess a structure resembling that
Next, the anomeric acetyl group of 1,2,3,4,6-penta-O-acetyl-D-
of glycogen instead of mimicking the sugar diphosphonucleo-
glucopyranoside (2) was selectively cleaved using benzylamine
tide donor molecule.
in THF at room temperature to furnish 3,30 which was
With this objective in mind, our goal was to design, syn-
employed for the glycosylation of commercially available
thesize and analyze a molecule that might potentially resemble
4-hydroxyazobenzene, by means of the Mitsunobu reaction.31
a short branch of glycogen and that additionally included a
The resulting 4-(phenylazo)phenyl-2,3,4,6-tetra-O-acetyl-D-
molecular switch, whose structure could be altered by an exter-
glucopyranoside (4) was deacetylated with MeONa/MeOH to
nal stimulus such as light. Azobenzene and spiropyran are the
give 4-(phenylazo)phenyl-D-glucopyranoside (1) in quantitative
most commonly used photoswitches in the design of mole-
yield.32,33 The azoglucoside 1 was obtained after four steps
cules with photomodulated biological activity.22–28 We have
with an overall yield of 34% as a mixture of the α and β
synthesized the glucoside 1 (Fig. 1) around the azobenzene
anomers. Integration of resonance of the anomeric proton in
photoswitch. Irradiation of the more stable (E)-form with UV
the 1H NMR spectrum of compound 1 showed that the α : β
light causes isomerization across the NvN double bond to the
ratio was approximately 1 : 4 (Fig. 1B).
bent (Z)-isomer, which by thermal equilibration or by exposure
to visible light relaxes back to the (E)-isomer. If the Z → E
Photochromic properties of the azobenzene glucoside 1
Fig. 2A shows the changes observed in the absorption spec-
trum of the azobenzene glucoside 1 after irradiation with UV
light in HEPES buffer, pH 7, at 30 °C. The spectrum of the
more stable (E)-isomer exhibits the typical bimodal absorption
profile of azobenzenes, which consists of a strong symmetry-
allowed π–π* transition with a maximum at 339 nm and
a weaker symmetry-forbidden n–π* transition centered at
ca. 430 nm. UV light induced E → Z photoisomerization of the
azobenzene glucoside causes a large decrease of the intensity
of the 339 nm band. Assuming that the (Z)-isomer does not
contribute to the absorption at this wavelength and that the
spectrum obtained before UV irradiation corresponds to 100%
of the (E)-isomer, the apparent fractional amount of the (Z)-
form present in the system, Yapp, can be determined by using
eqn (1):34
Fig. 1 (A) Synthesis of the azobenzene glucoside 1. (B) Partial 1H NMR
spectrum of 1 in D2O, showing the resonances of the anomeric protons
of the α and the β anomer and their relative integrals.
Aph
Yapp ¼ 1 ꢀ
ꢁ 100
ð1Þ
A0
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Biological activity of the azobenzene glucoside 1
The inhibitory potential of the azobenzene glucoside 1 on the
enzymes responsible for the degradation and synthesis of gly-
cogen was tested using purified preparations of commercially
available RMGPa and recombinantly produced EcGS, respecti-
vely. Recombinant SuSy 4 from potatoes was also included in
the study as a representative of a non-glycogen metabolizing
GT. RMGPa activity was assayed in the direction of phosphoro-
lysis and EcGS and SuSy 4 in the direction of synthesis of glyco-
gen and sucrose, respectively. All the assays were performed
using a saturating concentration of the respective substrates in
HEPES buffer, pH 7.0 (10 mM Pi and 4 mg mL−1 glycogen for
RMGPa; 2 mM ADPG and 4 mg mL−1 glycogen for EcGS; 5 mM
UDPG and 30 mM fructose for SuSy 4). A stock solution of
(E)-1 was serially diluted and each one of the dilutions was
irradiated for 20 minutes to reach the photostationary state. The
effect of either photoisomer of the azobenzene glucoside 1 as
the GT inhibitor was measured in a concentration-dependent
manner. Thereby, sigmoidal curves were obtained (Fig. 3A and
B), from which IC50 values for each isomer were deduced
(Fig. 3C). It must be noted that the actual IC50 values of the
(Z)-isomer must be somewhat lower than those shown in
Fig. 3C, since, for the sake of simplicity, calculations were
done assuming 100% photoconversion of the (E)-isomer into
the (Z)-form upon irradiation. This analysis revealed that the
ground state (E)-azobenzene glucoside is a moderate SuSy 4
inhibitor (IC50 = 0.73 mM), while the (Z)-form is found to be a
weak inhibitor, such that its IC50 could not be determined (not
shown). RMGPa was moderately inhibited by both photo-
isomers, with IC50 values of 4.9 mM and 2.4 mM for the
(E)- and (Z)-forms, respectively. In contrast, this initial evaluation
Fig. 2 (A) UV/Vis absorption spectrum of 1 (30 μM in HEPES buffer, pH
7, 30 °C) before (solid line) and after (dashed line) irradiation with UV
light (320 nm < λirrad < 390 nm) for 10 minutes. (B) Absorbance (339 nm)
versus time trace for the thermally-activated Z → E back reaction of a
solution of 1 (30 μM in HEPES buffer, pH 7, 30 °C), after the photo-
stationary state had been reached. The trace was fitted to a biexponen-
tial function (R2 = 0.99995), from which the values of the respective
amplitudes (A1 = 0.10
0.01; A2 = 0.42
0.01) and first order decay
constants (k1 = 1.57 0.02 × 10−4 s−1; k2 = 9.57 0.09 × 10−6 s−1) were
estimated.
where A0 and At stand for the initial absorbance (100%
(E)-isomer) and the absorbance after a given irradiation time,
respectively, both measured at 339 nm. In our system, the
photostationary state, in which no further change of the elec-
tronic spectrum was observed, was reached after approximately
10 minutes of irradiation. The apparent extent of E → Z photo-
isomerization for the azobenzene glucoside 1 was 75%, a value
lower than those previously reported for the isolated α- and
β-azobenzene glucosides, which were estimated to be higher
than 95%.35 It must be noted, however, that we have analyzed
the extent of photoconversion in aqueous solution whereas the
reported values were determined in DMSO.
When illumination ceased and the sample was kept in the
dark, the thermally-activated Z → E reaction led back to the
thermodynamically stable (E)-isomer. This process was fol-
lowed by monitoring the absorbance at 339 nm of a sample of
1 irradiated with UV light until the photostationary state was
reached and then kept in the dark at 30 °C (Fig. 2B). The
kinetic data obtained could be fitted to a biexponential func-
tion with a ratio of the amplitudes for the two distinct phases
of 1 : 4, which closely matches the α : β ratio obtained from the
1H NMR spectrum of compound 1. Thus, the data for the
smaller amplitude phase can be ascribed to the thermal Z → E
back isomerization of the α anomer, while the larger ampli-
tude phase derives from the more abundant β anomer of the
azobenzene glucoside 1. Both anomers showed slow Z → E
back isomerization reactions, with first order rate constants of
1.57 × 10−4 s−1 and 9.57 × 10−6 s−1, which correspond to half-
life times of 1.22 h and 20.1 h, for the α and β anomers,
respectively. Again, these half-life values are somewhat lower
than those estimated for the separate α- and β-azobenzene
glucosides in DMSO.35 Both the large extent of E → Z conversion
attained in the photostationary state and the long half-life
times of the generated (Z)-isomers allowed us to test the bio-
logical activity of these isomers without the need for a continu-
ous light source illuminating the sample.
Fig. 3 Relative RMGPa (A) and EcGS activities (B) at varying con-
□
●
centrations of the (Z) ( ) and (E) ( ) photoisomers of azobenzene
glucoside 1. IC50 values of (E)-1 and (Z)-1 for SuSy 4, RMGPa and EcGS
inhibition (C). *IC50 of (Z)-1 for SuSy 4 could not be determined, since
this form does not inhibit the enzyme to half of its initial value at the
highest concentration tested. Values are the mean of three independent
experiments.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
showed that while the (E)-form of 1 is a moderately strong (Ki = 352 32 μM) (Fig. 4F). Altogether, these results dictate
EcGS inhibitor (IC50 = 1.6 mM), the inhibitory potency of the that EcGS follows an ordered bi–bi kinetic mechanism, where
bent (Z)-isomer increased more than 50-fold (IC50 = 31.5 μM). ADPG binds first to the free enzyme, followed by glycogen that
Thus, the azobenzene glucoside 1 is an excellent photoswitch- binds to the ADPG–enzyme complex.36,37 Moreover, this
able selective inhibitor for the regulation of the EcGS activity kinetic analysis indicated that both isomers of the azobenzene
(Fig. 3C).
glucoside 1 mimic the acceptor substrate and occupy the glyco-
We next examined the kinetic mechanism of the inhibition gen subsite in the catalytic center of EcGS.
exerted by compound 1 on EcGS. The double reciprocal plots
of EcGS activity under pseudo single substrate conditions con-
verge to a negative value, which is characteristic of a sequential
binding mechanism36 of the two substrates, ADPG and glyco-
Docking of azobenzene glucoside 1 into the glycogen-binding
site of E. coli glycogen synthase
gen, excluding a ping-pong mechanism (Fig. 4A). In addition, Compound 1 is a 1 : 4 mixture of the α- and β-anomers. This
double reciprocal analyses in the presence of varying concen- raises the question as to which of the two anomers is the
trations of the inhibitor molecule (Fig. 4B–E) showed that the actual EcGS inhibitor. To try to answer this question and to
(Z)-form of 1 is a non-competitive inhibitor with respect to visualize complexation of the different forms of the azobenz-
both ADPG and glycogen, with observed inhibitory constants ene glucoside within the active site of GS, computer-aided
of 38 5 nM and 67 4 nM, respectively.
docking of these molecules to the available X-ray structure of
The (E)-isomer was also found to be a non-competitive EcGS was performed. The crystal structure of the EcGS mutant
inhibitor with respect to ADPG (Ki = 122 21 μM) and glycogen E377A in complex with ADPG and maltopentaose as an accep-
tor analogue of glycogen (PDB 3CX4)38 was employed for
docking the four geometric isomers of the azobenzene gluco-
side, α-(E), β-(E), α-(Z) and β-(Z), to the site normally occupied
by glycogen in the active site (Fig. 5). The structures of the four
isomers were previously optimized at the B3LYP/3-21G level of
theory.39 For these calculations, the (E)-isomer was character-
ized by an inversion angle α (the NvN–C angle) of 120° and a
rotation angle ω (the dihedral angle C–NvN–C describing a
rotation around the central NvN bond) of 180°. For the (Z)-
form the inversion angle α was fixed to 120° and the rotation
angle ω to 10°, since this dihedral angle at the NvN double
bond for azobenzene is experimentally found to range from 8
to 12°, using X-ray methods and magnetic resonance spectro-
scopy techniques.40,41
In the best docked conformation, only the β-(Z)-isomer of
compound 1 presents a good overlap of the glucosyl moiety
and the two aromatic rings with the first three glucose resi-
dues of the crystallographic ligand maltopentaose (Fig. 5C).
This analysis further shows that the C2 hydroxyl group of the
glucose residue is within the hydrogen bonding distance
(2.9 Å) with the side chain of Asp137 and the C6 hydroxyl
group with the backbone amide of Gly17 (2.8 Å). The aromatic
ring next to the glucose moiety is also well oriented to estab-
lish π-stacking interactions with the Tyr95 side chain (3.1 Å).
Finally, the second N of the NvN double bond can form a
2.9 Å hydrogen bond with the side chain of His96. In the
remaining three geometric isomers, the aromatic ring distal to
the glucose unit resides at a position distant from that occu-
pied by the third glucose moiety of the bound maltopentaose
and they present several unfavorable interactions with various
amino acid residues of the glycogen-binding site. Thus,
docking analysis provides a reasonable explanation for the dis-
Fig. 4 Double reciprocal plot of EcGS activity under single substrate
conditions (A). Double reciprocal analyses of EcGS activity in the pres-
ence of varying concentrations of the (Z)-isomer (B and C) or (E)-isomer
(D and E) of azobenzene glucoside 1. Inhibition constants of (E)-1 and
tinct inhibitory potency exhibited by the isomers of compound
1 on the EcGS activity and allows us to propose that the more
abundant β-anomer with the (Z)-configuration of the azobenz-
ene moiety is the actual inhibitor since it is the molecule that
(Z)-1 on EcGS activity (F). Values are the mean of three independent
experiments.
most resembles a bound glycogen molecule.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 5 Computer modelled docking of the azobenzene glucoside 1 to the glycogen binding site of EcGS. The figure shows the α-(E) (A, in grey),
β-(E) (B, in pink), α-(Z) (C, in orange) and β-(Z) (D, in green) forms of compound 1. Selected EcGS amino acids are shown in blue and three glucose
residues from the bound maltopentaose and ADP are shown in yellow.
Conclusions
Experimental section
Kinetic parameters of the photoinduced E → Z and thermal
Z → E isomerization of the azobenzene glucoside 1
We have designed and synthesized the azobenzene glucoside
1, which in its β-(Z)-form structurally resembles three terminal
glucose residues of a glycogen branch. The molecule incorpor- Solutions of the azobenzene glucoside 1 ca. 3 × 10−5 M in 1 cm
ates an azobenzene photoswitch whose conformation can be optical path quartz cells were irradiated with a Philips high-
significantly altered by irradiation with UV light. The large pressure mercury lamp (500 W nominal power) using a 0.5 M
attainable extent of photoconversion of compound 1 from the solution of Co(NO3)2 as an optical filter. Irradiation was
ground state (E)-isomer to the bent (Z)-form, and the long life pursued until no changes were observed in the electronic spec-
of the latter allowed us to analyze the inhibitory properties of trum of the sample. Afterwards the solution was thermostated
each photoisomer separately.
in the dark at the desired temperature and the thermal Z → E
While both isomers were modest inhibitors of RMGPa, and isomerization was monitored by the change in the electronic
only the (E)-form moderately inhibited SuSy 4, inhibitory spectrum of the sample. The observed rate constants and
potency in front of EcGS increased by more than 50 fold upon phase amplitudes were derived by fitting a biexponential func-
irradiation, and the (Z)-isomer showed an IC50 in the µM range tion to the absorbance at 339 nm versus time trace, using stan-
and kinetic inhibitory constants in the nM range. To the best dard software packages.
of our knowledge, the azobenzene glucoside 1 is, as the kinetic
and the docking analyses indicate, the first GS inhibitor that
mimics the acceptor substrate of the enzyme, namely glycogen.
Cloning, expression and purification of SuSy 1 from Solanum
tuberosum L
Although compound 1 was synthesized as a 1 : 4 mixture of the The full length cDNA encoding for potato SuSy 4 cloned into
α- and β-anomers, docking analysis suggests that the more the pET28a vector (Novagen) was kindly provided by Prof.
abundant β-anomer is the one responsible for the observed Javier Pozueta-Romero, from Instituto de Agrobiotecnología y
inhibition. These observations, and the fact that compound 1 Recursos Naturales, Universidad Pública de Navarra/CSIC,
conformation can be photomodulated, open up the possibility Spain. This construct attaches a 6xHis tag sequence to the
of further development of drugs that act selectively on glyco- N-terminus of SuSy 4. E. coli BL21(DE3) cells harboring
gen metabolic enzymes and whose biological effects can be the pET28a/SuSy 4 plasmid were grown at 37 °C up to an
spatially and temporally resolved.
OD600 ∼0.8 in LB medium supplemented with kanamycin
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
(30 μg mL−1). At this point, 0.8 mM IPTG was added and the NADPH in an assay coupled to phosphoglucomutase and
induction was carried out at 22 °C for 16–18 h. The cells were glucose-6-phosphate dehydrogenase as described by Schinzel
collected by centrifugation and resuspended in 50 mM Tris- and Palm44 with a slight modification. The enzyme activity was
HCl buffer (pH 7.4) plus 1 mM PMSF. Cell disruption was assayed at 30 °C in a 200 mM HEPES buffer (pH 7.0) contain-
carried out by sonication of the ice-cooled suspension, after ing NADP+ (2.2 mM), MgCl2 (1 mM), glucose 1,6-bisphosphate
which the insoluble material was separated by centrifugation (5 μM), glucose-6-phosphate dehydrogenase (1 unit), phospho-
at 13 000g for 45 min. The supernatant was loaded onto a glucomutase (1 unit), 10 mM NaH2PO4 and 4 mg mL−1 glyco-
HisTrap column (GE Healthcare), previously equilibrated with gen, and an appropriate amount of the enzyme. The reaction
50 mM Tris-HCl, pH 7.4. Recombinant SuSy 4 was eluted with was started by the addition of the enzyme and the increase in
a linear gradient of 0–250 mM imidazole in the same buffer absorbance at a wavelength of 340 nm, due to the formation of
and loaded on a Superdex 200 gel filtration column (GE NADPH, was followed continuously using a Bio-Rad Bench-
Healthcare). The eluted sample was analyzed by SDS-PAGE, mark Plus Microplate Spectrophotometer for 60 min at 30 °C.
and the fractions that contained a homogeneous enzyme, as One unit of enzyme activity is defined as the amount of
determined by SDS-PAGE, were pooled, concentrated with a enzyme catalyzing the formation of 1 μmol of glucose-1-phos-
Centriprep YM-30 (Millipore) and stored at −80 °C in 50 mM phate in 1 min at 30 °C.
Tris-HCl (pH 7.4) containing 10% (v/v) of glycerol.
Enzyme kinetic analysis
Plots of initial rates (μM s−1) versus substrate concentration
were fitted to the Michaelis–Menten equation by nonlinear
least squares regression using OriginPro 8.0. Alternatively, data
were linearized and fitted to a straight line by representing the
inverses of the initial rates versus inverses of the substrate
concentration.
Expression and purification of EcGS
BL21(DE3) cells harboring the pET-23a/EcGS plasmid42 were
grown at 37 °C in LB medium containing ampicillin (50 μg
mL−1) up to an optical density of ∼0.8 at 600 nm. At this point,
0.8 mM IPTG was added and the culture was grown at 23 °C
for an additional 14–16 h. Cells were collected by centrifu-
gation and resuspended in 50 mM Tris-HCl buffer (pH 7.4)
plus 1 mM PMSF. Cell disruption was carried out by sonication
of the ice-cooled suspension. The insoluble material was sep-
Docking studies
The structures of (E)- and (Z)-isomers of the azobenzene gluco-
side 1 were optimized at the B3LYP/3-21G level of theory using
Gaussian 03W version 6.0. The coordinates and refinement
restraint files for both (E)-1 and (Z)-1 were prepared using
the PRODRG of CCP4 program suite 6.3.0. Computer-aided
docking of the optimized structures was performed by using
Coot 0.7.0. Docking studies were based on the X-ray structure
of the E. coli glycogen synthase (PDB 3CX4).38
arated by centrifugation at 13 000g for 45 min, and the super-
natant was loaded into a nickel affinity column (HisTrap HP,
GE Healthcare). The column was washed with 50 mM Tris-HCl
(pH 7.4) and His-tagged EcGS was eluted with a linear gradient
of 0–1 M imidazole and loaded on a Superdex 200 gel filtration
column (GE Healthcare). The fractions that contained a homo-
geneous enzyme, as determined by SDS-PAGE, were pooled,
concentrated with a Centriprep YM-30 (Millipore) and stored
at −80 °C in 50 mM Tris-HCl (pH 7.4), containing 10% (v/v) of
glycerol.
Assay of SuSy 4 and EcGS activity. Sucrose and glycogen
synthase activities were measured at 30 °C using the method
reported by Morell and Copeland43 in which the UDP or ADP,
respectively, produced by the enzymes in the synthesis direc-
tion is coupled to NADH oxidation via pyruvate kinase and
lactate dehydrogenase. For SuSy activity measurements the
reaction mixture (100 µL) contained 200 mM HEPES buffer
(pH 7.0), 30 mM fructose, 5 mM UDPG, 0.7 mM phosphoenol-
pyruvic acid, 0.6 mM NADH, 50 mM KCl, 13 mM MgCl2, pyru-
vate kinase (7.5 U), lactate dehydrogenase (15 U) and an
appropriate amount of the enzyme. The same reaction mixture
was used for EcGS, except that 30 mM fructose and 5 mM
UDPG were replaced with 4 mg mL−1 glycogen and 2 mM
ADPG, respectively. One unit of enzyme activity is defined as
the amount of the enzyme producing 1 μmol of UDP or ADP in
1 min at 30 °C.
Acknowledgements
This work was supported by the Dirección General de Investi-
gación, Ministerio de Ciencia y Tecnología, Spain, grants
CTQ2006-00743 (to J. C. F.), BFU2011-30554 (to J. J. G.), and
CTQ2012-36074 (to D. V.) and the CIBER de Diabetes y Enfer-
medades Metabólicas. M. Díaz-Lobo is grateful for the doctoral
grant BES-2009-029354 from the Ministerio de Ciencia e
Investigación. J. Garcia-Amorós is grateful for a Beatriu de
Pinós postdoctoral grant from the Generalitat de Catalunya
(Spain, 2011 BP-A-00270).
Notes and references
1 S. Ghosh, D. Usharani, A. Paul, S. De, E. D. Jemmis and
S. Bhattacharya, Bioconjugate Chem., 2008, 19, 2332–2345.
2 F. Tanaka, T. Mochizuki, X. Liang, H. Asanuma, S. Tanaka,
K. Suzuki, S. Kitamura, A. Nishikawa, K. Ui-Tei and
M. Hagiya, Nano Lett., 2010, 10, 3560–3565.
Assay of rabbit muscle glycogen phosphorylase activity
Glycogen phosphorylase activity was measured in the direction
of glycogen degradation (phosphorolysis) by determination of
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
3 D. G. Flint, J. R. Kumita, O. S. Smart and G. A. Woolley, 22 C. Amato, A. Fissi, L. Vaccari, E. Balestreri, O. Pieroni and
Chem. Biol., 2002, 9, 391–397. R. Felicioli, J. Photochem. Photobiol., B, 1995, 28, 71–75.
4 J. Levitz, C. Pantoja, B. Gaub, H. Janovjak, A. Reiner, 23 M. Aizawa, K. Namba and S. Suzuki, Arch. Biochem.
A. Hoagland, D. Schoppik, B. Kane, P. Stawski, A. F. Schier, Biophys., 1977, 180, 41–48.
D. Trauner and E. Y. Isacoff, Nat. Neurosci., 2013, 16, 507– 24 I. Willner, S. Rubin and A. Riklin, J. Am. Chem. Soc., 1991,
516. 113, 3321–3325.
5 H. Asanuma, X. Liang, T. Yoshida, A. Yamazawa and 25 Y. Hu, R. F. Tabor and B. L. Wilkinson, Org. Biomol. Chem.,
M. Komiyama, Angew. Chem., Int. Ed., 2000, 39, 1316–1318. 2015, 13, 2216–2225.
6 A. Yamazawa, X. Liang, H. Asanuma and M. Komiyama, 26 A. Estévez-Torres, C. Crozatier, A. Diguet, T. Hara, H. Saito,
Angew. Chem., Int. Ed., 2000, 39, 2356–2357.
7 V. Borisenko, D. C. Burns, Z. H. Zhang and G. A. Woolley,
J. Am. Chem. Soc., 2000, 122, 6364–6370.
8 P. Osman, S. Martin, D. Milojevic, C. Tansey and
F. Separovic, Langmuir, 1998, 14, 4238–4242.
K. Yoshikawa and D. Baigl, Proc. Natl. Acad. Sci. U. S. A.,
2009, 106, 12219.
27 W. A. Velema, J. P. Van der Berg, M. J. Hansen,
W. Szymanski, A. J. Driessen and B. L. Feringa, Nat. Chem.,
2013, 5, 924–928.
9 Z. Haiqian, S. Hiroaki, G. Ning, S. Hiroshi and 28 O. Srinivas, N. Mitra, A. Surolia and N. Jayaraman, J. Am.
S. Masahiko, J. Southeast Univ., 2001, 17, 22–26. Chem. Soc., 2002, 124, 2124–2125.
10 R. Behrendt, C. Renner, M. Schenk, F. Q. Wang, 29 H. G. Sudibya, J. Ma, X. Dong, S. Ng, L. J. Li, X. W. Liu and
J. Wachtveitl, D. Oesterhelt and L. Moroder, Angew. Chem.,
Int. Ed., 1999, 38, 2771–2774.
11 S. Sporlein, H. Carstens, H. Satzger, C. Renner,
R. Behrendt, L. Moroder, P. Tavan, W. Zinth and
J. Wachtveitl, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 7998–
8002.
P. Chen, Angew. Chem., Int. Ed., 2009, 48, 2723–2726.
30 S. A. Allman, H. H. Jensen, B. Vijayakrishnan, J. A. Garnett,
E. Leon, Y. Liu, D. C. Anthony, N. R. Sibson, T. Feizi,
S. Matthews and B. G. Davis, ChemBioChem, 2009, 10,
2522–2529.
31 O. Mitsunobu, Synthesis, 1981, 1–28.
12 I. Willner and S. Rubin, Angew. Chem., Int. Ed. Engl., 1996, 32 C. D. Hurd and R. P. Zelinski, J. Am. Chem. Soc., 1947, 69,
35, 367–385. 243–246.
13 A. Cattani-Scholz, C. Renner, C. Cabrele, R. Behrendt, 33 N. Laurent, D. Lafont, F. Dumoulin, P. Boullanger,
D. Oesterhelt and L. Moroder, Angew. Chem., Int. Ed., 2002,
41, 289–291.
G. Mackenzie, P. H. J. Kouwer and J. W. Goodby, J. Am.
Chem. Soc., 2003, 125, 15499–15506.
14 D. A. James, D. C. Burns and G. A. Woolley, Protein Eng., 34 H. Dürr and H. Bouas-Laurent, in Studies in Organic Chem-
2001, 14, 983–991. istry 40, Elsevier, Amsterdam, 1990, p. 1068.
15 J. H. Harvey and D. Trauner, ChemBioChem, 2008, 9, 191– 35 V. Chandrasekaran, E. Johannes, H. Kobarg, F. D. Sçnnichsen
193. and T. K. Lindhorst, ChemistryOpen, 2014, 3, 99–108.
16 M. R. Banghart, A. Mourot, D. L. Fortin, J. Z. Yao, 36 H. Bisswanger, Enzyme Kinetics: Principles and Methods,
R. H. Kramer and D. Trauner, Angew. Chem., Int. Ed., 2009,
48, 9097.
WILEY-VCH Verlag GmbH & Co. KGaA, Weinhem, 2008,
p. 301.
17 A. Mourot, T. Fehrentz, Y. Le Freuvre, C. M. Smith, 37 W. E. Schäfer, J. M. Rohwer and F. C. Botha, Eur. J. Biochem.,
C. Herold, D. Dalkara, F. Nagy, D. Trauner and
R. H. Kramer, Nat. Methods, 2012, 9, 396–402.
18 P. Stawski, M. Sumser and D. Trauner, Angew. Chem., Int.
Ed., 2012, 51, 5748–5751.
2004, 271, 3971–3977.
38 F. Sheng, A. Yep, L. Feng, J. Preiss and J. H. Geiger, Bio-
chemistry, 2009, 48, 10089–10097.
39 E. G. Lewars, Springer Science and Business Media, 2010.
19 E. Van Dijkum, R. Danac, D. J. Hughes, R. Wood, A. Rees, 40 A. Mostad and C. Romming, Acta Chem. Scand., 1971, 25,
B. L. Wilkinson and A. J. Fairbanks, Org. Biomol. Chem.,
2009, 7, 1097–1105.
20 J. C. Errey, S. S. Lee, R. P. Gibson, C. Martinez Fleites,
C. S. Barry, P. M. J. Jung, A. C. O’Sullivan, B. G. Davis and
G. J. Davies, Angew. Chem., Int. Ed., 2010, 49, 1234–1237.
21 P. Peltier, M. Beláňová, P. Dianišková, R. Zhou,
3561–3568.
41 J. Dokić, M. Gothe, J. Wirth, M. V. Peters, J. Schwarz,
S. Hecht and P. Saalfrank, J. Phys. Chem. A, 2009, 113,
6763–6773.
42 A. Díaz, M. Díaz-Lobo, I. Fita, J. J. Guinovart and
J. C. Ferrer, IUBMB Life, 2012, 64, 649–658.
R. B. Zheng, J. A. Pearcey, M. Joe, P. J. Brennan, C. Nugier- 43 M. Morell and L. Copeland, Plant Physiol., 1985, 78, 149–
Chauvin, V. Ferrières, T. L. Lowary, R. Daniellou and
154.
K. Mikušová, Chem. Biol., 2010, 17, 1356–1366.
44 R. Schinzel and D. Palm, Biochemistry, 1990, 29, 9956–9962.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.