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Biochem. J. (2012) 441, 881–887 (Printed in Great Britain)
881
doi:10.1042/BJ20110721
Modulation of glucokinase by glucose, small-molecule activator and
glucokinase regulatory protein: steady-state kinetic and cell-based analysis
Francis J. BOURBONAIS, Jing CHEN, Cong HUANG, Yanwei ZHANG, Jeffrey A. PFEFFERKORN and James A. LANDRO1
Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
GK (glucokinase) is an enzyme central to glucose metabolism
that displays positive co-operativity to substrate glucose. Smallmolecule GKAs (GK activators) modulate GK catalytic activity
and glucose affinity and are currently being pursued as a treatment
for Type 2 diabetes. GK progress curves monitoring product
formation are linear up to 1 mM glucose, but biphasic at 5 mM,
with the transition from the lower initial velocity to the higher
steady-state velocity being described by the rate constant kact . In
the presence of a liver-specific GKA (compound A), progress
curves at 1 mM glucose are similar to those at 5 mM, reflecting
activation of GK by compound A. We show that GKRP (GK
regulatory protein) is a slow tight-binding inhibitor of GK.
Analysis of progress curves indicate that this inhibition is time
dependent, with apparent initial and final Ki values being 113 and
12.8 nM respectively. When GK is pre-incubated with glucose
and compound A, the inhibition observed by GKRP is time
dependent, but independent of GKRP concentration, reflecting
the GKA-controlled transition between closed and open GK
conformations. These data are supported by cell-based imaging
data from primary rat hepatocytes. This work characterizes the
modulation of GK by a novel GKA that may enable the design of
new and improved GKAs.
INTRODUCTION
genome-wide association studies support a linkage between
GKRP polymorphisms and serum-fasting plasma glucose levels
[13,14]. Physiologically, the association between GK and GKRP
is enhanced by F-6-P (fructose 6-phosphate) and diminished by
F-1-P (fructose 1-phosphate) [15], with evidence suggesting that
both sugar phosphates bind to the same site on the regulatory
protein [15]. In addition, GKRP associates with GK in a manner
that is competitive with glucose [16], and there is evidence that
GKRP is a tight-binding inhibitor of GK [17].
A model based on crystallographic evidence has been proposed
to account for the kinetic co-operative behaviour of GK. The
model assumes two distinct slowly interconverting conformations
induced by a major reorientation following ligand binding. The
first conformation is an inactive super-open form with low affinity
for glucose, whereas the second is an active closed form with a
higher affinity for glucose [18]. When glucose binds to the higheraffinity lower-abundance conformer, equilibration increases the
proportion of the higher affinity form [19,20]. Pre-steady-state
kinetic analysis supports this model [21]. Further refinements
demonstrate a landscape of conformations available between the
extremes of the forms super-open and closed the forms as well as
the existence of a GK–GKA complex in the absence of glucose
[22]. Therefore both kinetic and structural studies support the
hypothesis that multiple interconverting conformations of GK
exist with discrete rate-limiting domain reorganizations. This
model allows one to kinetically explore GK modulation by
glucose, small-molecule activators and GKRP.
In the present study, we show with steady-state kinetic methods
that there are at least two kinetically distinct forms of GK that
interconvert through a slow conformational change and that this
interconversion is affected by glucose concentration and a liverspecific GKA (J. A. Pfefferkorn, A. Guzman-Perez, J. Litchfield,
R. Aiello, J. L. Treadway, J. Pettersen, M. L. Minich, K. J. Filipski,
GK (glucokinase; hexokinase IV) plays a central role in
maintaining glucose homoeostasis and is the major glucosephosphorylating enzyme expressed in hepatocytes and pancreatic
β-cells [1]. GK is unique among hexokinases in that it displays
a sigmoidal substrate dose–response curve, demonstrates low
affinity and positive co-operativity for substrate glucose, and
is not susceptible to product (glucose 6-phosphate) inhibition
[2,3]. These properties are critical to the role GK plays as
the glucose sensor. Given its pivotal role in regulating glucose
homoeostasis, there has been significant interest in GK as a
target for treating T2DM (Type 2 diabetes mellitus). A number
of small-molecule GKAs (GK activators) have been discovered
that allosterically enhance GK activity [4]. In vitro, these GKAs
increase GK activity by enhancing the apparent affinity for
glucose and/or increasing the turnover rate (kcat ) of the enzyme
[5]. In vivo studies revealed that GKAs were able to decrease
glucose levels in normal and diabetic animal models [4,6–8].
However, although early-generation dual-acting (liver and
pancreas) GKAs were efficacious, many were also reported to
have significant hypoglycaemia risks that have been attributed, in
part, to increasing insulin secretion at inappropriately low glucose
levels. As an alternative, hepatoselective activators may avoid
this hypoglycaemia liability, albeit while potentially offering less
efficacy [9]. Hepatic GK activity, unlike pancreatic GK activity, is
regulated by an endogenous 68 kDa regulatory protein inhibitor
called the GKRP (GK regulatory protein) [10]. Under conditions
of limited nutrients, GKRP associates with and sequesters GK
in the nucleus. Under nutrient-rich conditions, the GK/GKRP
complex dissociates and GK translocates to the cytoplasm [11,12].
There is evidence from rodent models of T2DM that this
translocation is impaired in the disease state [12] and human
Key words: allosteric modulation, glucokinase activator (GKA),
glucokinase regulatory protein (GKRP), liver-selective, slow,
tight-binding inhibition.
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; F-1-P, fructose 1-phosphate; F-6-P, fructose 6-phosphate;
GK, glucokinase; GKA, GK activator; GKRP, GK regulatory protein; G6PDH, glucose-6-phosphate dehydrogenase; OATP, organic anion transporting
polypeptide; T2DM, Type 2 diabetes mellitus.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
882
Figure 1
F. J. Bourbonais and others
Compound A
C. S. Jones, M. Tu, G. Aspnes, H. Risley, S. W. Wright, J. Bian,
B. Stevens, P. Bourassa, T. D’Aquila, L. Baker, N. Barucci, A.
S. Robertson, F. Bourbonais, D. R. Derksen, M. MacDougall,
C. Over, J. Chen, A. L. Lapworth, J. A. Landro, K. Atkinson,
N. Haddish-Berhane, B. Tan, L. Yao, R. E. Kosa, M. V. Varma,
B. Feng, D. B. Duignan, A. El-Kattan, S. Murdande, S. Liu, M.
Ammirati, J. Knafels, P. DaSilva-Jardine, L. Sweet, L. Spiros and
T. P. Rolph, unpublished work). We demonstrate that GKRP is a
slow tight-binding inhibitor of GK, and explore the modulation
of this mechanism with a GKA. Corroborative modulation of GK
by glucose and a GKA is demonstrated using a cellular imagebased assay. A detailed understanding of the regulation of GK
in the liver by GKRP in the presence of a GKA is of particular
importance for understanding the effects of small-molecule GK
activation in the liver and for the design of new GKAs.
with minor modifications. All enzyme assays were carried out in
50 mM Hepes, pH 7.1, 10 mM MgCl2 , 25 mM KCl, 2.5 mM ATP,
1.1 mM NADP, 0.1 % BSA, 2 mM DTT and 1 unit/ml G6PDH
at 25 ◦ C in a final volume of 40 μl. GKA stock solutions were
prepared in 100 % DMSO and the final concentration of DMSO
in the assay never exceeded 1 %. All reactions were carried out
in a 384-well microtitre plate (Corning 3702) and the data were
collected continuously at 340 nm on a Spectramax 384 Plus.
Kinetic studies
To study the kinetics of GK activation, various concentrations
of glucose or glucose plus compound A were added to GK in a
glucose-free assay buffer. Data acquisition was initiated within
15 s and the reaction was monitored continuously every 10 s for
time periods up to 15 min. To study the kinetics of GK inhibition
by GKRP, various concentrations of GKRP or GKRP plus F-6-P
were added to GK in assay buffer containing 20 mM glucose or
20 mM glucose and a GKA. Data acquisition was initiated within
15 s and the reaction was monitored continuously every 15 s for
time periods up to 30 min. All reactions were run in triplicate or
more and results are means +
− S.D.
Analysis of the steady-state kinetics of GK inhibition by GKRP
EXPERIMENTAL
General
The steady-state initial velocities obtained from the preincubation studies with GK and various concentrations of GKRP
were analysed according to eqn (1) of Williams and Morrison
[28]
Compound A, (S)-6-{3-cyclopentyl-2-[4-(trifluoromethyl)-1Himidazol-1-yl]propanamido}nicotinic acid (Figure 1), was
synthesized using a method, for which details are available from
J.A.P. on request (email [email protected]).
G6PDH (glucose-6-phosphate dehydrogenase), insulin, dexamethasone M2 anti-FLAG resin and hexokinase I from
Saccharomyces cerevisiae were purchased from Sigma. Collagencoated plates were from BD Biosciences. Hoechst 33342, FBS
and all DMEM (Dulbecco’s modified Eagle’s medium) were
purchased from Invitrogen. Alexa Fluor® 594-labelled goat antirabbit antibody was purchased from Santa Cruz Biotechnology.
All other chemicals and reagents were purchased from various
commercial sources at the highest level of purity available. All
data were analysed with the indicated equations using GraphPad
Prism. Statistical analysis was carried out using SAS 9.3 software.
where ET is the total concentration of active enzyme, I T is the
total concentration of inhibitor, K i app is the apparent dissociation
constant of inhibitor, v is the initial velocity of the reaction in
the presence of inhibitor at concentration [I] and v0 is the initial
velocity of the reaction in the absence of inhibitor. For the case
of substrate and inhibitor competing for the same enzyme active
site, Ki app is related to the true dissociation constant Ki by the
relationship shown in eqn (2):
Protein expression and purification
K iapp = K i (1 + [S]/K m )
Recombinant human β-cell N-terminally hexahistidine-tagged
GK was purified from Escherichia coli as described previously
[24]. The purified protein was stored in 25 mM Hepes, 150 mM
NaCl, 5 mM DTT (dithiothreitol) and 5 % (v/v) glycerol pH 7.5 at
− 80 ◦ C. Recombinant human FLAG-tagged GKRP was cloned
as described in [25] and expressed in and purified from Sf9
insect cells using baculovirus-infected cell technology [26].
Cells were harvested 72 h post-infection and protein purified
using affinity column chromatography (M2 anti-FLAG resin)
followed by size-exclusion column chromatography (Superdex
200). Purified GKRP was stored in 25 mM Tris/HCl, pH 7.5,
150 mM NaCl, 10 % (v/v) glycerol and 2 mM TCEP [tris-(2carboxyethyl)phosphine] at − 80 ◦ C.
Enzyme assay
GK activity was measured by monitoring the rate of glucose 6phosphate formation using the G6PDH/NADP-coupled assay [27]
c The Authors Journal compilation c 2012 Biochemical Society
v
/v0 =
1−
([E T ] − [IT ] − K iapp ) +
app
app
([E T ] − [IT ] − K i )2 + 4[E T ]K i
2[E T ]
(1)
(2)
where K m is the Michaelis constant and [S] is the competing
concentration of the substrate.
Analysis of GK activation and inhibition kinetics
Progress curve analysis allowed for the determination of the firstorder rate constant (k) for the transition of GK from the initial
rate (v0 ) to the steady-state rate (vs ). The progress curves for both
activation and inhibition of GK were analysed by eqn (3) [29]:
(v0 − vs )
(1 − e−kt )
(3)
k
where product p represents the absorbance at 340 nm at any time
t, v0 and vs are the initial and steady-state velocities of the reaction
in the presence of the inhibitor respectively, and k is the rate of
the transition from v0 to vs .
The derived rate constants (k) for the inhibition of GK with
varying amounts of GKRP, with and without F-6-P, were further
p = vs t +
Steady-state modulation of glucokinase
analysed using eqns (4)–(6) to determine Ki . For the case of
tight-binding inhibition, the concentration of the EI complex
is not negligible in comparison with the concentration of I, so
the assumption that free and total inhibitor concentrations are
equivalent is not valid. Eqn (4) allows for calculation of [EI] for
a tight-binding inhibitor:
√
(E T + IT + K i ) (E T + IT + K i )2 − 4E T IT
(4)
[EI] =
2
where [EI] is the concentration of enzyme–inhibitor complex,
I T is the total concentration of inhibitor and ET is the total
concentration of enzyme. The relationship between the rate of
transition from initial to final velocity described above (k) and the
kinetic constants for the forward rate of formation of the final,
tight EI complex (kf ) and the reverse rate back to the initial EI
complex (kr ), is given by eqn (5):
k = kr +
k f IF
K iapp + IF
(5)
where I F is the concentration of free inhibitor calculated from the
relationship I F = I T − EI and K i app is as described above. Global
fitting of data to eqns (4) and (5) yields the best-fit values for kf
and kr . The relationship between the initial velocity K i ’ and the
steady-state velocity K i is given by eqn (6):
kf
Ki = Ki
(6)
kf + kr
GK translocation assay
The translocation of GK from the nucleus to cytoplasm in
primary hepatocytes was performed as described in [30] with
minor modifications. Cryopreserved hepatocytes from Wistar
rats were plated into collagen-coated 96-well plates (60 000
cells/well) in DMEM containing FBS, 100 nM insulin, 10 nM
dexamathasone and 2 mM sodium pyruvate. Following 4 h of
incubation in a humidified incubator at 37 ◦ C and 10 % CO2 ,
fresh medium containing Matrigel was added to the cells and
they were incubated overnight. The medium was changed to
DMEM (no glucose) with 10 % FBS, 100 nM insulin, 10 nM
dexamethasone, 2 mM L-glutamine and the cells were incubated
for 12–16 h. Medium was aspirated and glucose at concentrations
of 2.5 or 8.9 mM was added to DMEM containing 0.07 % BSA,
1 nM insulin and 10 nM dexamethasone. Compound A was added
to the cells at concentrations ranging from 0.3 nM to 100 μM.
Following a 1-h incubation at 37 ◦ C and 10 % CO2 , cells were
fixed with 8 % (v/v) paraformaldehyde for 15 min followed by
4 % paraformaldehyde for 30 min at 4 ◦ C. Cells were washed
with PBS and permeablized using 0.1 % Triton X-100 in PBS
for 20 min at room temperature (23 ◦ C). Following washing with
PBS, cells were treated with 50 μl of blocking solution (3 % BSA
and 1 % goat serum in PBS) and incubated at room temperature
for 1 h. An anti-GK antibody (diluted 1:250 in blocking solution)
was added to the cells and incubated at 4 ◦ C overnight. Cells were
washed and incubated with an AF594-conjugated goat anti-rabbit
antibody (diluted 1:500) with 2 μM Hoechst in PBS at room
temperature for 1 h.
Image analysis for GK translocation
Array Scan (Cellomics, Thermo Fisher Scientific) imaging was
performed using an λex of 364 nm for the Hoechst 33342 dye and
an λem of 594 nm for the AF594/GK signal. Images were analysed
using a compartmental analysis algorithm to determine the GK
883
distribution between the nucleus and cytoplasm. Compound
response (y) was defined as change in pixel intensity in the red
GK signal channel between the nucleus and cytoplasm relative
to the values at different glucose concentrations (2.8 or 8.9 mM)
in the extracellular medium. GraphPad Prism was then used to fit
dose–response curves to eqn (7):
f (x) = ylow + (yhigh − ylow )/[1 + 10(log EC50 −x)×Hill ]
(7)
where x is the concentration of the compound, y is the compound
response and Hill indicates the Hill coefficient.
RESULTS
Observation of the assay transient
The previous work has demonstrated that at least two distinct
forms of GK exist and interconvert via a substrate-induced
conformational change as observed using standard kinetic
methods [20]. We wanted to confirm this observation and
determine the effect of a GKA on the phenomenon. By monitoring
GK progress curves after GK was transferred from a glucosefree assay buffer to a buffer containing glucose, we were able to
observe a lag in product formation where the initial velocity v0 was
lower than the steady-state velocity vs (Figure 2). The transition
time from the initial to the steady-state velocity was dependent
on the concentration of glucose, and the transition rate constant
kact could be calculated from eqn (3). For glucose concentrations
up to 1 mM, the progress curves were linear for 5 min with no
observed difference between the initial and steady-state velocities
(Figure 2A, left-hand y-axis). For glucose concentrations above
1 mM, there was a measurable difference between the initial and
steady-state velocities, and this was reflected in the value of kact
(Figure 2A, right-hand y-axis and Table 1). At 5 mM glucose, the
−1
value of kact was 0.025 +
− 0.001 s and remained constant up to
50 mM glucose (results not shown).
The transition of GK from initial to steady-state velocity was
also measured in the presence of a novel GKA, compound A
(J. A. Pfefferkorn, A. Guzman-Perez, J. Litchfield, R. Aiello, J.
L. Treadway, J. Pettersen, M. L. Minich, K. J. Filipski, C. S.
Jones, M. Tu, G. Aspnes, H. Risley, S. W. Wright, J. Bian, B.
Stevens, P. Bourassa, T. D’Aquila, L. Baker, N. Barucci, A. S.
Robertson, F. Bourbonais, D. R. Derksen, M. MacDougall, C.
Over, J. Chen, A. L. Lapworth, J. A. Landro, K. Atkinson, N.
Haddish-Berhane, B. Tan, L. Yao, R. E. Kosa, M. V. Varma,
B. Feng, D. B. Duignan, A. El-Kattan, S. Murdande, S. Liu,
M. Ammirati, J. Knafels, P. DaSilva-Jardine, L. Sweet, L.
Spiros and T. P. Rolph, unpublished work). Compound A is a
potent and selective activator optimized for recognition by liver
specific OATPs (organic anion transporting polypeptides). This
activator has enhanced hepatic uptake, low hepatic oxidative
metabolism and low passive permeability to minimize distribution
into peripheral tissues that lack OATP transporters such as
pancreas. Biochemically, compound A demonstrated in vitro
activation of human recombinant GK with an EC50 of 90 nM.
Moreover, compound A shifted the S0.5 for glucose from 13
to 5.9 mM, and increased the V max 1.5-fold at concentration
of 2.3-fold EC50 (i.e. 210 nM). In the present study, when
tested at 210 nM, there was a noticeable difference between the
initial and steady-state velocities for all glucose concentrations
tested. The value of kact measured at 1.0 mM glucose was
−1
0.01 +
at 5 mM glucose, the value of kact
− 0.002 s , whereas
−1
was 0.006 +
0.0007
s
,
on
average 3.6 times larger than in the
−
absence of activator. The progress curves for 1 and 5 mM glucose
are shown (Figure 2A, left- and right-hand y-axis respectively).
c The Authors Journal compilation c 2012 Biochemical Society
884
F. J. Bourbonais and others
Effect of compound A on time dependence of GK inhibition by GKRP
Figure 2 Observation of the GK assay transient as a function of glucose
and compound
(A) Representative GK progress curves in the absence and presence of compound A at 1 mM
(left-hand y -axis; 75 nM GK) and 5 mM (right-hand y -axis; 15 nM GK) glucose. The reactions
were initiated with glucose and ATP, and the data were collected every 10 s for 5 min for
conditions (䊐) 1 mM glucose, (䊊) 5 mM glucose, (䊏) 1 mM glucose plus 210 nM compound
A; (䊉) 5 mM glucose plus 210 nM compound A. See the text for details. (B) Comparison of GK
progress curves in (A) (䊉) where the reaction is initiated by the addition of 5 mM glucose, ATP
and compound A compared with pre-incubation of GK with 5 mM glucose and compound A for
20 min, followed by initiation of the reaction with ATP (䉱). This comparison indicates that the
lag in the progress curves observed when GK is not pre-incubated with glucose or glucose and
compound A is not due to an artefact of the coupling system.
Values for v0 , vs and kact for 1, 3, 5 and 10 mM glucose are shown
in Table 1. Two experiments support that the observation of the
transient is not due to an artefact of the assay system. When
GK is pre-incubated with compound A and glucose (5 mM), there
is no observable difference between the initial and steady-state
velocities, which is distinct from the observation when there is
no pre-incubation step (Figure 2B). Secondly, no transient
is observed under any conditions when hexokinase family
members I, II or III, enzymes that do not display co-operative
behaviour with substrate, are substituted for GK. It should be
noted that the value of kact , although influenced by the on-rate of
compound A for GK, was not dominated by this event (results not
shown).
Table 1
GKRP is an endogenous inhibitor of GK in hepatocytes. Previous
work has demonstrated that GKRP inhibits GK in a manner that
is competitive with glucose [16] that the association between
GK and GKRP is enhanced by F-6-P [15] and that GKRP
inhibits GK in a tight-binding manner [17]. Given the tightbinding nature of the inhibition of GK by GKRP, we investigated
the time dependence of the interaction. Preliminary experiments
indicated that GK inhibition by GKRP was time dependent and
slow enough to be measured under standard steady-state assay
conditions. Experiments monitoring GK inhibition by GKRP
as a function of time in the absence and presence of 210 nM
compound A were performed after a 20 min pre-incubation of
GK with glucose, DMSO vehicle or compound A, and F-6-P. The
assay was initiated by the addition of standard assay reagents plus
various concentrations of GKRP, and had a final concentration of
100 μM F-6-P. The data clearly show that the inhibition was
dependent on time and GKRP concentration for experiments
performed without and with compound A (Figures 3A and
3B respectively). In the absence of compound A, non-linear
regression analysis of progress curves using eqn (3) showed that
the apparent rate constant describing the time dependence for the
transition from initial velocity v0 to steady-state velocity vs , kobs ,
increased with GKRP concentration. When kobs was plotted as
a function of GKRP concentration, the data followed a biphasic
hyperbolic function (Figure 3C, closed squares), indicating that a
fast equilibrium preceded a final slow-dissociating complex thus
supporting a two-step tight inhibition mechanism. When these
data were analysed with eqns (5) and (6), the values of kr and kf
were 0.0014 +
− 0.0002 and 0.011 +
− 0.003/s respectively, and the
steady-state K i value reported was 12.8 nM, whereas the initial K i
value (K i in eqn 6) was 113 nM. The steady-state value of
K i was similar to the value of 3.1 +
− 0.2 nM determined by a preincubation of GK (40 nM) with variable GKRP concentrations
for 20 min in the presence of 500 μM F-6-P, and analysis of the
data using eqns (1) and (2). When compound A was included in
the pre-incubation mixture, time-dependent inhibition was still
observed, but the value of kobs did not appear to vary with GKRP
concentration (Figure 3C, closed circles), suggesting the approach
to vs was governed by a conformational change reflecting the
transition of GK from a compound A-induced closed form to
the open form. Statistical analysis of these data was performed
using both a linear model and ANOVA. When GKRP
concentration was treated as a continuous variable, a linear
model between the kobs value and the independent variable GKRP
suggested that kobs was not likely to be linearly associated with
GKRP concentration (the P value of the significance testing for
the slope was 0.1031). When GKRP concentration was treated as
categorical variable, ANOVA of kobs with GKRP concentration
as fixed effect suggested that kobs was not likely constant
across GKRP concentrations (the P value of the significance
Values of v 0 , v s and k act with or without compound A at various glucose concentrations
N/A, not applicable.
v 0 (nmol · mg − 1 · s − 1 )
v S (nmol · mg − 1 · s − 1 )
k act (s − 1 )
Glucose (mM)
DMSO
Compound A
DMSO
Compound A
DMSO
Compound A
1
3
5
10
4.6 +
− 0.3
7+
−3
17 +
−2
27 +
− 4.5
4+
−2
7.5 +
−2
21 +
−2
29 +
− 11
4.6 +
− 0.3
25 +
−1
63 +
−3
180 +
− 10
56 +
−2
130 +
−8
456 +
−7
564 +
− 10
N/A
0.024 +
− 0.001
0.025 +
− 0.0009
0.024 +
− 0.003
0.011 +
− 0.0016
0.007 +
− 0.0007
0.006 +
− 0.0007
0.007 +
− 0.0008
c The Authors Journal compilation c 2012 Biochemical Society
Steady-state modulation of glucokinase
885
Figure 4 Effect of compound A on GK translocation from the nucleus to the
cytoplasm in cryopreserved hepatocytes from Wistar rats
(A) Comparison of the effects of 2.5 mM glucose (black bar), 8.9 mM (white bar) glucose
and 8.9 mM glucose plus 100 μM compound A (checkered bar) on translocation. Nucleus
to cytoplasm intensity refers to GK intensity differences between nucleus and cytoplasm.
(B) A dose-dependent effect of compound A at 2.5 mM (䊏) or 8.9 mM (䉱) glucose on
the translocation of GK from the nucleus to the cytoplasm was observed in cryopreserved
hepatocytes from Wistar rats. Compound response values were calculated with respect to each
glucose/vehicle control. Results are means +
− S.D. (n = 3).
Figure 3
Progress curves for the inhibition of GK by GKRP
(A) Inhibition in the absence of compound A. The reaction mixture contained 10 nM GK, 20 mM
glucose, 100 μM F-6-P and GKRP at zero (䊉), 15 nM (䊏), 30 nM (䉱) and 60 nM (䉲). GK
and glucose were pre-incubated for 20 min and the reaction was initiated with the addition of
GKRP and ATP to 2.5 mM. (B) Effect of compound A on time-dependent GKRP inhibition. The
reaction was performed as in (A) except compound A was present in the pre-incubation with
glucose at a concentration of 210 nM and GKRP was present at zero (䊉), 30 nM (䊏), 100 nM
(䉱) and 300 nM (䉲). (C) Dependence of k obs on GKRP concentration in the absence (䊏) or
presence (䊉) of compound A. The GK progress curves from (A) and (B) were analysed with eqn
(3) to obtain k obs as a function of GKRP concentration. Note that in addition to the concentrations
shown in (A) and (B), concentrations of GKRP ranging from 15 to 150 nM were tested.
testing for the fixed effect of levels of GKRP was <0.0001).
Although there exist statistically significant differences between
some concentrations of GKRP in terms of the kobs value, those
differences do not appear to be a function of GKRP concentration.
Cell-based GK translocation assay
A cell-based assay was used to translate our biochemical
observations of the effect of compound A on the modulation
of GK activity by GKRP to a more physiological setting. The
translocation of GK from the nucleus to the cytoplasm was
measured in primary rat hepatocytes and monitored by high-
content image analysis [30]. In primary rat hepatocytes cultured
under low glucose concentrations, GKRP retained GK in the
nucleus in an inhibited state. In the presence of increased glucose
concentration, the interaction between GK and GKRP was destabilized; GK was subsequently released and translocated to the
cytoplasm, where the active conformer phosphorylated glucose. In
addition to glucose concentration, the effect of compound A was
tested in this assay. As shown in Figure 4(A), increasing glucose
concentration caused a reduction in the nucleus to cytoplasm GK
intensity, reflecting the translocation of GK from the nucleus to
cytosol. Compound A further increased the cytoplasmic shift of
GK in a dose–dependent manner. As shown in Figure 4(B), the
EC50 value decreased with an increasing glucose concentration,
from an EC50 of 1.46 μM at 2.5 mM glucose to 0.26 μM at
8.9 mM glucose.
DISCUSSION
The unique properties of GK, a monomeric protein displaying
positive co-operativity with respect to glucose, permits
responsiveness to glucose in the relevant physiological range
of 2.5–5.0 mM glucose observed in the normal glycaemic state
and over a broader range (7.0–15 mM) under conditions of
hyperglycaemia characteristic of T2DM. Although a number
of models have been proposed to account for experimental
observations, the ‘mnemonical’ model [31], which has been
c The Authors Journal compilation c 2012 Biochemical Society
886
F. J. Bourbonais and others
further refined as the pre-existing equilibrium model [21],
appears to be supported by a wealth of experimental
information [22]. In the simplest scenario, GK exists in two
forms with different glucose-binding affinities to give the
same kinetically indistinguishable enzyme–glucose complex. As
glucose concentration increases, the co-operativity of glucose
binding is reflected in an equilibrium shift from a higher
abundance lower glucose affinity form E to a higher affinity form
E*. The lower-affinity form was observed in our experiments
(Figure 2) at concentrations of glucose up to 1 mM, as evidenced
by the linear GK progress curve. As glucose concentration
increased beyond 1 mM, the transition from the low-affinity form
to the high-affinity form was reflected in the steady-state transition
from v0 to vs . The transition kact , which we observed by our steadystate method, reflected a combination of glucose binding and
kinetic constants describing conformational changes from E to E*.
The addition of compound A alters the GK affinity for glucose and
the kinetic rate constants describing the conformational changes
leading to GK activation. We showed that compound A increases
the steady-state velocity of GK at the lowest concentration of
glucose tested (1 mM), by driving the equilibrium towards a
higher activity state. At 1 mM glucose, the v0 to vs transition
was measurable and the value of kact was found to be 0.01 s − 1 . We
also observed an increase in transition time of approximately 4fold at the higher glucose concentrations tested. Several potential
models may explain the increased transition time: compound
A may simply interfere with the transition from E to E*
along the glucose-induced conformation path; compound A may
direct the transition from E to E* along an alternate conformational pathway; or compound A may induce an alternative energy
state of GK that is distinct from E*. Given the observable
transition from v0 to vs at 1 mM glucose, the increased glucose
affinity, the increased V max , and longer transition time from v0 to
vs for glucose concentrations at or above 3 mM, we propose that
compound A induces an alternative GK conformation E** that is
distinct from E*. It is noteworthy that compound A activates GK
at glucose concentrations that are considerably lower than what
are considered euglycaemic. However, given that compound A
is largely confined to the liver, the possibility of hypoglycaemia
resulting from pancreatic insulin secretion is minimized.
The interaction between GK and GKRP may shed light
on the how compound A influences GK conformation and
enzyme activity. Compound A did not alter the affinity between
GK and GKRP; however, compound A increased GK activity
by 3.5-fold relative to the control in the presence of GKRP,
but interestingly in the absence of GKRP, compound A only
increased the activity of GK by 1.5-fold over glucose alone
(results not shown). This increased relative activation of GK
by compound A in the presence compared with the absence
of GKRP suggests that in the presence of GKRP, a second
mechanism may be operative. Glucose is competitive with GKRP
for binding to GK and we report that compound A increased
the affinity of GK for glucose. It follows that in the presence
of compound A, less GK would be bound to GKRP, creating a
larger active GK population. This phenomenon was confirmed in
rat hepatocytes when GK compartmentalization was monitored
using cell imaging technology, where compound A was shown
to potentiate the action of glucose in disrupting the interaction
between GK and GKRP and promoted the translocation of GK
from nucleus to cytosol. We also observed that in the presence of
compound A, the time-dependent inhibition of GK by GKRP was
no longer dependent on GKRP concentration. This observation
suggests that the rate-limiting step of the inhibition may be
governed by an E** to E* transition. Alternatively, compound A
may simply drive the equilibrium from E towards the closed more
c The Authors Journal compilation c 2012 Biochemical Society
active GK conformer E*. Although plausible, this explanation
does not account for the changes in glucose affinity, increases in
GK activity in response to compound A, and does not account for
the effect compound A has on the value of kact . A third possibility is
that by binding to the allosteric site on GK, compound A may slow
the transition from E* back to E. This mechanism is consistent
with the increase in transition time from v0 to vs , but does not
account for the increased affinity for glucose nor does it explain
the increase in enzyme activity. Therefore a transition from E**
to E* is the likely rate determining step in the time-dependent
inhibition of GK by GKRP in the presence of compound A.
In summary, our observations about the effects of GKAs on
GK kinetics in the presence and absence of GKRP may, in part,
enable the further characterization of GKAs and the relationship
between activation and pharmacological effects.
AUTHOR CONTRIBUTION
All authors contributed to the writing of the paper. In addition, specific contributions are
as follows: Frank Bourbonais and Cong Huang performed the biochemical experiments
and made intellectual contributions to the design and interpretation of those experiments.
Jing Chen performed the cell-based translocation experiments and made intellectual
contributions to the design and interpretation of those experiments. Yanwei Zhang
performed a statistical analysis and interpretation of the data appearing in Figure 3(C).
Jeffrey Pfefferkorn is the project leader for the Pfizer Glucokinase programme and made
intellectual contributions to various aspects of the work. James Landro made intellectual
contributions to all aspects of the work appearing in the paper.
ACKNOWLEDGEMENTS
We thank Paul DaSilva-Jardine, Ann Aulabaugh, Dave Beebe, Jessica Ward and Rich
Derksen for a critical reading of the paper.
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Received 21 April 2011/20 October 2011; accepted 1 November 2011
Published as BJ Immediate Publication 1 November 2011, doi:10.1042/BJ20110721
c The Authors Journal compilation c 2012 Biochemical Society