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INTRODUCTION |
Glucokinase (hexokinase D or IV), an enzyme playing the role of a
glucose sensor in
-cells of pancreatic islets and in liver (reviewed
in Refs. 1 and 2), displays a sigmoidal saturation curve for its hexose
substrate (3-5). This property, which sensitizes the enzyme to changes
in the blood glucose level, cannot be accounted for by classical models
of cooperativity (6, 7), because glucokinase is a monomer (8).
Sigmoidal behavior due to random, nonequilibrium addition of the two
substrates, glucose and ATP-Mg (9), can be excluded on the basis that
there is no inhibition by ATP (10). Models that better account for the
kinetic behavior of glucokinase assume the existence of two different
conformations with different affinities for glucose that interconvert
slowly (11, 12), thus allowing glucose to increase the proportion of
the conformation with higher affinity. These kinetic models are
supported (a) by the observation that cooperativity is lost when the rate of the reaction is slow, due to the use of a poor nucleotide substrate (13) or to the presence of a low ATP concentration and an inhibitory ADP concentration (11); (b) by the
identification of slow kinetic transients (14); and (c) by
the demonstration of slow conformational changes by fluorescence
spectroscopy (15).
The possibility that the cooperativity is due to the existence of two
binding sites for glucose, with regulatory and catalytic function,
respectively, is usually ruled out (16) on the basis of experiments
showing that glucose protects glucokinase against inactivation by a
substrate analog,
N-(N-bromoacetyl)-6-aminohexanoylglucosamine, with an hyperbolic instead of sigmoidal dependence on concentration (17). However, Agius and Stubbs (18) recently observed that mannoheptulose, a competitive inhibitor of glucokinase, is more effective to decrease the Hill coefficient of this enzyme than other
inhibitors such as N-acetylglucosamine or 5-thioglucose. This led them to postulate that mannoheptulose inhibits glucokinase by
binding to a putative allosteric site for glucose.
Fructose is known to be a poor substrate for glucokinase, displaying no
(12, 19) or positive (14, 20) cooperativity for this enzyme.
Remarkably, fructose phosphorylation was shown to be markedly
stimulated by glucose and, to a lesser extent, by mannose (19). The
stimulation of fructose phosphorylation by glucose and its analogs
could therefore be a simple way of probing the specificity of a
putative allosteric site.
In the present work we have further characterized the effect of glucose
and its analogs on the phosphorylation of fructose by wild type
glucokinase and by point mutants that have a reduced cooperativity or
affinity for glucose. As the use of [14C]fructose in a
radiochemical assay of glucokinase is hampered by the low affinity of
the enzyme for this substrate, we have developed an enzymatic assay
involving sorbitol-6-phosphate dehydrogenase, which allows the specific
measurement of fructose 6-phosphate without interference by glucose
6-phosphate. Our results indicate that the stimulation exerted by
glucose on the phosphorylation of fructose is mediated by binding of
glucose to the catalytic site.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemicals were from Sigma or Merck.
D-Fructose was from Acros. The recombinant
Xenopus regulatory protein of glucokinase was purified up to
the DEAE-Sepharose step (21). The recombinant human islet glucokinase
was purified as described previously (22). The preparation,
purification, and characterization of human
-cell glucokinase
mutants have been described by Veiga-da-Cunha et al. (23)
for N166R, D158A, and V203A/D158A and by Moukil et al. (22)
for Y214A and C230S.
Preparation of Escherichia coli Sorbitol-6-phosphate
Dehydrogenase--
The open reading frame of E. coli
sorbitol-6-phosphate dehydrogenase (24) was amplified by polymerase
chain reaction using genomic DNA from strain JM109 as a template
and oligonucleotide primers with the following sequences: upstream
primer, 5'-ggcgccatatgaatcaggttggcgttgtc-3'; downstream primer,
5'-cgctggatccatcagaacatcacctgaccg-3'. The
upstream primer incorporates the starting codon (bold) in the NdeI restriction site (underlined), whereas
the downstream primer incorporates a BamHI restriction site
(underlined) which flanks the termination codon
(bold). The ~0.8-kilobase pair amplified fragment was
inserted in pET3a, and the resulting plasmid was used to overexpress
sorbitol-6-phosphate dehydrogenase in Bl21pLysS (25). Expression was
carried out in 1 liter of ZB medium at 19 °C for 40 h in
the presence of 0.4 mM isopropylthiogalactoside. The cells
were pelleted by centrifugation, resuspended in 45 ml of homogenization
buffer (25 mM Hepes, pH 7.1, 5 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml
antipain, 0.5 mM phenylmethylsulfonyl fluoride), and
extracted in a French pressure cell. The extract was centrifuged at
15,000 × g for 30 min, and an ammonium sulfate
fraction (15-30% of saturation) was prepared from the resulting
supernatant. This fraction was resuspended in 2 ml of Buffer A (20 mM Hepes, pH 7.1, 1 mM dithiothreitol) and
chromatographed on a 1.6 × 70-cm Sephacryl S-200 HR column equilibrated with Buffer A. The active fractions were pooled and chromatographed on a 1.6 × 13-cm DEAE-Sepharose equilibrated in Buffer A supplemented with 10 mM NaCl; the column was
washed with the same buffer, and protein was eluted with a linear NaCl
gradient (10-500 mM in 2 × 50 ml of Buffer A). About
5000 units of >95% homogeneous sorbitol-6-phosphate dehydrogenase
could be obtained in this way. The enzyme had a specific activity of
~200 µmol/min/mg. It was stored at 4 °C as a precipitate in 70%
ammonium sulfate.
Enzyme Assays--
In the course of its purification,
sorbitol-6-phosphate dehydrogenase was assayed at 30 °C through the
change in A340 in a reaction mixture containing
50 mM Hepes, pH 7.1, 2 mM fructose 6-phosphate,
and 0.15 mM NADH.
Glucokinase was assayed spectrophotometrically at 30 °C in 1 ml of
assay mixtures containing, unless otherwise indicated, 25 mM Hepes, pH 7.1, 2 mM ATP-Mg, 5 mM
MgCl2, 25 mM KCl, 1 mM dithiothreitol, and the indicated concentrations of substrates and
effectors. Fructose 6-phosphate production was measured in the presence
of 0.15 mM NADH and 2 units of sorbitol-6-phosphate dehydrogenase; glucose 6-phosphate production, in the presence of 0.5 mM NAD+ and 10 µg/ml Leuconostoc
mesenteroides glucose-6-phosphate dehydrogenase; and ADP
formation, with 0.15 mM NADH, 0.25 mM
phosphoenolpyruvate, 10 µg/ml pyruvate kinase, and 10 µg/ml lactate
dehydrogenase. Results shown are means of at least three
determinations ± S.E.
Other Methods--
Protein concentration was measured according
to Bradford (26) using bovine serum albumin as a standard.
Modelization--
To simplify the writing of equations derived
from the model shown in Fig. 7, the concentrations of E, EG, E', and
E'G are called "A," "B," "C," and "D," and the
concentration of glucose is termed "G." Kinetic constants are
symbolized by letters (a-h and m). Note that the product (a.g.d.f) is
necessarily equal to (b.h.c.e). A system of four equations with 4 unknowns can be written as follows.
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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In Equation 1, the sum is actually equal to a constant, which,
for the sake of simplicity, is assumed to be equal to 1. Equations 2-4
are derived from the consideration that, under steady state conditions,
C, D, and B remain constant.
A fifth equation allows one to calculate the rate of product
formation.
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(Eq. 5)
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Values are assigned to the kinetic constants (see Fig. 7). The
system of Equations 1-4 was solved by matrix calculus, and
v was calculated for discrete concentrations of glucose
using Microsoft Excel, Version 1998. Hill coefficients were calculated
for substrate concentrations corresponding to ~25% of
Vmax.
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RESULTS |
Purification of Sorbitol-6-phosphate Dehydrogenase and Its Use in
the Assay of Glucokinase--
To monitor specifically the formation of
fructose 6-phosphate without interference of glucose 6-phosphate, we
decided to use sorbitol-6-phosphate dehydrogenase. This bacterial
enzyme catalyzes the reversible NADH-dependent conversion
of fructose 6-phosphate to sorbitol 6-phosphate (24). The open reading
frame encoding E. coli sorbitol-6-phosphate dehydrogenase
(24) was inserted into a pET3a expression vector (25) and used to
overexpress the enzyme. An activity of ~40 µmol/min/mg protein was
observed in extracts of cells induced with
isopropyl-1-thio-
-D-galactopyranoside as compared
with 0.25 µmol/min/mg protein in cells that did not harbor the plasmid.
The enzyme was purified by chromatography on Sephacryl S-200 and
DEAE-Sepharose. SDS polyacrylamide gel electrophoresis indicated that
the preparation contained one single band with the expected size (26 kDa). The preparation was free from enzymes such as hexokinase and
phosphoglucose isomerase, which could potentially interfere in the
glucokinase assay. The Km for fructose 6-phosphate was 0.38 mM and that for NADH was 28 µM.
Using this enzyme, we determined that, at pH 7.0, the thermodynamic
equilibrium of the reaction [NAD+][sorbitol
6-phosphate]/[NADH][fructose 6-phosphate] amounted to 252 ± 17.
In the presence of this enzyme and of NADH, glucokinase could be
assayed with fructose as a substrate through the decrease in
A340. Similar rates were observed with this
assay as with others in which the formation of fructose 6-phosphate is
monitored through the reduction of NAD+ to NADH in the
presence of phosphoglucose isomerase and glucose-6-phosphate dehydrogenase or in which the formation of ADP is measured through the
oxidation of NADH in the presence of phosphoenolpyruvate, pyruvate
kinase, and lactate dehydrogenase (not shown).
Saturation Curve for Fructose and Effect of Glucose--
Fig.
1 shows that human islet glucokinase
displayed a positive cooperativity for fructose with an
S0.5 of 580 mM and a Hill coefficient of about
1.5. Glucose suppressed this cooperativity and, at low concentrations,
stimulated the enzyme, with S0.5 decreasing to about 170 mM in the presence of 4 mM glucose. At more
elevated concentrations, glucose exerted competitive inhibition.

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Fig. 1.
Hill plot of the saturation curve of
human -cell glucokinase by fructose; effect of
glucose. Glucokinase activity was measured through the formation
of fructose 6-phosphate in the presence of the indicated concentrations
of fructose and glucose. Glucose had no effect on the
Vmax value of the enzyme, which amounted to 183 µmol/min/mg.
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As some authors (12, 19) but not others (14, 20) reported a hyperbolic
saturation curve for fructose, we also studied the fructose saturation
curve under the conditions described by Cardenas et al. (12)
but found sigmoidal kinetics similar to those shown in Fig. 1. We
checked also enzymatically (27) that the preparation of fructose that
we used was not detectably contaminated by glucose (<0.01%). We
therefore have no explanation for this discrepancy.
The effect of glucose on fructose phosphorylation was further
investigated in the experiment shown in Fig.
2 in which the rates of both fructose and
glucose phosphorylation have been recorded. Fructose phosphorylation
was stimulated by a factor of up to about 6 by 5 mM glucose
(Fig. 2A). By contrast, glucose phosphorylation was not
stimulated by fructose, but rather inhibited, particularly at the
highest concentrations of glucose used (Fig. 2B).
Remarkably, the increment in the rate of fructose phosphorylation
induced by glucose amounted in some cases to much more than the rate of glucose phosphorylation. Thus at 200 mM fructose, the
increment of fructose phosphorylation induced by 1 mM
glucose represented ~18 µmol/min/mg as compared with a rate of
glucose phosphorylation of ~1.5 µmol/min/mg.

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Fig. 2.
Effect of glucose on fructose phosphorylation
(A) and of fructose on glucose phosphorylation
(B) by human -cell
glucokinase. The formations of fructose 6-phosphate (A)
and glucose 6-phosphate (B) were measured in the presence of
the indicated concentrations of fructose and glucose.
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Suppression of the Stimulatory Effect of Glucose with Poor
Phosphoryl Donors--
It was previously shown that the cooperativity
for glucose is lost when ATP is substituted by a poor phosphoryl donor
such as ITP (13) or when the concentration of ATP is low, most
particularly in the presence of ADP, which acts as a competitive
inhibitor versus ATP (11). Fig.
3 shows that the stimulation of the
phosphorylation of 20 mM fructose by 5 mM
glucose is about 6-fold in the presence of 0.4 to 5 mM ATP
and that it is less than 2-fold in the presence of nucleotides that are
poor substrates (ITP, CTP, GTP, and UTP) or when the enzyme is measured
in the presence of a low concentration of ATP and an inhibitory
concentration of ADP. There is a clear hyperbolic relationship between
the rate of fructose phosphorylation in the absence of glucose and the
relative stimulation exerted by glucose.

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Fig. 3.
Effect of the nature and the concentration of
nucleotides on the stimulation of fructose phosphorylation by
glucose. Glucokinase activity was measured through the formation
of fructose 6-phosphate at 20 mM fructose in the absence or
in the presence of 5 mM glucose with the indicated
concentrations of nucleotides (in mM). The graph
shows the relationship between the stimulation exerted by glucose on
fructose phosphorylation and the rate of fructose phosphorylation
measured in the absence of glucose. For the sake of clarity,
error bars are shown only for some points.
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Glucose Analogs Acting as Stimulators or Inhibitors of Fructose
Phosphorylation--
Among a series of analogs that we tested, only
three could stimulate fructose phosphorylation, mannose,
2-deoxyglucose, and glucosamine (Fig. 4).
These compounds are all substrates for glucokinase, and their capacity
to stimulate fructose phosphorylation (Fig. 4A) correlated
with their ability to be substrate (Fig. 4B), mannose, and
2-deoxyglucose displaying, respectively, about 2- and 5-fold higher
S0.5 values with Hill coefficients of 1.6 and 1.1. As
expected, these substrates acted as inhibitors of fructose
phosphorylation at more elevated concentrations.

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Fig. 4.
Effect of glucose analogs on the
phosphorylation of fructose (A) and comparison with
their ability to act as substrates (B). In
A glucokinase activity was measured through the formation of
fructose 6-phosphate at 100 mM fructose in the presence of
the indicated concentrations of hexoses. In B ADP formation
was measured in the presence of the indicated substrate, without
fructose.
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Other glucose analogs did not act as stimulators but only as inhibitors
of fructose phosphorylation (Fig. 5 and
not shown). These were either poor substrates of the enzyme
(2-fluoro-2-deoxyglucose (Km = 50 mM,
Vmax = 70% of the Vmax
with glucose) and mannoheptulose (Km = 4 mM, Vmax = 0.2%, IC50 = 9 mM at 100 mM fructose) or pure inhibitors
(5-thioglucose; IC50 = 2 mM), N-acetylglucosamine (IC50 = 2 mM),
N-acetylmannosamine (IC50 = 60 mM),
N-benzoylglucosamine (IC50 = 0.85 mM), and D-xylose (IC50 > 600 mM). The effect of some of these inhibitors was also tested in the presence of 5 mM glucose, which stimulated the
enzyme about 4-fold. When activities were expressed relative to the
activity measured in the absence of inhibitor, it appears that the
apparent affinity for the inhibitors that are structural analogs of
glucose was increased in the presence of glucose, whereas the
inhibition exerted by other competitive inhibitors (Xenopus
regulatory protein (21) and acyl-CoAs (28, 29)) was decreased in the
presence of glucose (Fig. 5).

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Fig. 5.
Effect of glucose (0 or 5 mM) on
the inhibition of the phosphorylation of fructose (100 mM) by N-acetylglucosamine, mannoheptulose,
N-benzoylglucosamine, 5-thioglucose, palmitoyl-CoA, and the
regulatory protein. The glucokinase activity was measured through
the formation of fructose 6-phosphate at 100 mM fructose in
the absence ( ) or in the presence ( ) of 5 mM glucose
with the indicated concentrations of inhibitors. The 100% value
corresponds to ~11 µmol/min/mg in the absence of glucose and ~44
µmol/min/mg in the presence of 5 mM glucose.
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Other compounds had no effect on the phosphorylation of fructose either
in the presence or in the absence of glucose. These include (with the
maximal concentration tested shown in parentheses) 1,5-anhydro-D-sorbitol (50 mM),
-methyl-D-glucoside (300 mM), 1,5-anhydro-D-mannitol (50 mM),
D-allose (50 mM), D-galactose (250 mM), L-sorbose (600 mM),
6-deoxy-D-glucose (10 mM), and
D-glucuronamide (100 mM) (not shown).
Studies on Glucokinase Mutants--
To further investigate whether
the stimulation exerted by glucose was linked to the positive
cooperativity for this substrate, we investigated this effect in two
point mutants (N166R and Y214A) that have been produced by substituting
a glucokinase-specific residue with a residue in an equivalent position
in hexokinases with low Km values. These mutants
have a reduced Hill coefficient and an increased affinity for glucose
without marked change in Vmax (23, 22). The
saturation curves for fructose were almost hyperbolic (Hill
coefficients
1.2), and the S0.5 values equated 150 and 120 mM, respectively (not shown). As shown in Fig.
6A, glucose exerted a modest,
maximally 1.6-fold stimulatory effect in these mutants as compared with
the control (up to 4-fold).

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Fig. 6.
Effect of glucose on the phosphorylation of
fructose by mutants with low cooperativity, Y214A and N166R
(A), or by mutants of the catalytic site, C230S and
V203A/D158A (B). Glucokinase activity was
measured through the formation of fructose 6-phosphate in the presence
of fructose concentrations yielding ~15% of the
Vmax value observed with glucose as substrate.
They were 100 (wild type), 20 (Y214A), 30 (N166R), 400 (C230S), 30 (D158A), and 500 mM (V203A/D158A). Specific activities were
11, 21.3, 6, 12, and 7 µmol/min/mg for wild type, Y214A, N166R,
C230S, D158A, and V203A/D158A, respectively.
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We have also investigated the effect of glucose on fructose
phosphorylation in two glucokinase mutants in which a residue that is
next to the glucose-binding residue Asn-204 or Asn-231 had been
substituted (30). These mutants are V203A (which is combined with the
incidental mutation D158A) and C230S. In each case, fructose
phosphorylation was tested at a concentration corresponding to ~15%
of the Vmax value observed with glucose as
substrate. As shown in Fig. 6B, these two mutations
increased the concentration of glucose required to half-maximally
stimulate fructose phosphorylation by 8- and 2-fold, respectively,
whereas the S0.5 values for glucose were increased by 12- and 2-fold (23, 22), respectively, compared with the appropriate controls.
Simulation of a Slow Transition Model--
Because the results
obtained did not indicate the existence of a second binding site for
glucose, we decided to check whether a slow transition model could
account for the kinetic properties of glucokinase, including the
cooperativity for glucose and for fructose, the effect of glucose on
fructose phosphorylation, the effect of mutations such as Y214A that
reduce the Hill coefficient while increasing the apparent affinity for
glucose, and the better ability of mannoheptulose as compared with
N-acetylglucosamine to suppress cooperativity.
To achieve this goal, we performed mathematical simulations of the
model shown in Fig. 7, in which, for the
sake of simplicity, the binding of ATP and the release of the two
products ADP and glucose 6-phosphate are combined into one single
irreversible step. Kinetic constants values (Fig. 7, see
table) were chosen so that (a) the
interconversion between the two states was orders of magnitude slower
than the binding of the substrate(s) or the formation of products and
(b) state E' had 3750-fold more affinity for glucose than
state E. These values have been adjusted on a trial and error basis to
mimic more closely the kinetic behavior of glucokinase.

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Fig. 7.
Model for glucokinase. Free glucokinase
would exist under two interconvertible forms with low (E) or
high (E') affinity for glucose. Kinetic constants are
symbolized with lowercase letters (a-h and
m). Capital letters (A-D) symbolize
the concentrations of the indicated species. The table shows
the value of constants used in the text or for Figs. 8 and 9.
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The set of parameters shown in Fig. 7 predicted an S0.5
value of 8.4 mM for glucose and of 460 mM for
fructose, with Hill coefficients of ~1.7 and 1.4, respectively, close
to the measured values. The effect of a mutation such as Y214A on the
glucose saturation curve could be easily simulated by increasing the
E'/E ratio at equilibrium by a factor of 100. This decreased the
S0.5 value for glucose to 1.5 mM and the Hill
coefficient to 1.16 (as compared with measured values of 1.3 mM and 1.2, respectively; see Ref. 22).
The model also simulated the activation exerted by glucose on the
phosphorylation of fructose. As observed experimentally, the increase
in the rate of fructose phosphorylation was under some conditions
"more than stoichiometric" with respect to the rate of glucose
phosphorylation, and glucose phosphorylation was not detectably
stimulated by fructose (Fig. 8). As
expected, loss of cooperativity for glucose was observed when the rate
of the catalytic step was reduced (e.g. a Hill
coefficient of 1.2 was obtained when kinetic constant m was
decreased from 100 to 3 s
1; not shown).

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Fig. 8.
Simulation of the reciprocal effect of
glucose and fructose on their phosphorylation by glucokinase. The
constants used are given in Fig. 7.
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The model could also account for the differential behavior of
N-acetylglucosamine and mannoheptulose, if one assumed that the difference between the affinities of the two states for
N-acetylglucosamine was lower (by a factor of 100 in the
simulation shown in Fig. 7, the E' form having still 37.5-fold more
affinity for N-acetylglucosamine than the E form) than for
glucose, whereas it was the same for mannoheptulose as for glucose.
Fig. 9 shows a plot of the Hill coefficient versus the S0.5 values for glucose
in the presence of increasing concentrations of the two inhibitors. The
curves corresponding to the values obtained by simulation are close to the experimental values reported by Agius and Stubbs (18).

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Fig. 9.
Simulation of the effect of
N-acetylglucosamine and mannoheptulose on the
S0.5 and the Hill coefficient for glucose.
Closed symbols indicate the values obtained by Agius and
Stubbs (18), and open symbols indicate the values obtained
by mathematical simulation using constants shown in Fig. 7.
S0.5 and Hill coefficients were calculated for 0.2, 0.5, 1, 2, 5, and 10 mM mannoheptulose and for 0, 0.1, 0.2, 0.5, 1, 2, and 5 mM N-acetylglucosamine.
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DISCUSSION |
Lack of Evidence for an Allosteric Glucose-binding Site--
The
results presented in this work extend the previous observations of
Scruel et al. (19) that glucose stimulates the
phosphorylation of fructose. We show in addition in this paper that
this effect is largely reduced in two mutants (N166R and Y214A) that
have a reduced cooperativity for glucose. Furthermore, the stimulation is also decreased when the activity of the wild type enzyme is reduced
by replacing ATP by poorer phosphoryl donors, or when the ATP
concentration is reduced, most particularly in the presence of ADP.
Such a situation is known to decrease the Hill coefficient of the
glucose saturation curve to ~1.0 (11, 13). These observations indicate that the stimulation of fructose phosphorylation by glucose reflects the positive cooperativity for this substrate.
Therefore, if a potential allosteric glucose-binding site existed in
glucokinase as suggested by Agius and Stubbs (18), stimulation of
fructose phosphorylation would be a good means to characterize it. In
the present work, two pieces of evidence indicate, however, that such a
site does not exist. A first one is the fact that two mutants that have
a reduced affinity for glucose as a substrate, because of mutation of
residues in the catalytic site next to glucose binding residues Asn-204
and Asn-231, also have a reduced affinity for glucose as a stimulator
of fructose phosphorylation. Mutations affecting residues that directly
interact with glucose (Asn-204, Asp-205, Glu-256, and Glu-290) are not informative in this respect, because they lead to proteins that have
markedly decreased affinity for glucose and for other hexose substrates
and that do not display sigmoidal kinetics (31).
The second new piece of evidence is that, among a series of sugars,
only four (glucose, mannose, glucosamine, and 2-deoxyglucose), all
substrates of glucokinase, were able to stimulate the phosphorylation of fructose. If glucokinase had an independent (allosteric) binding site for glucose that would favor the binding of glucose to the catalytic site, one would expect the existence of glucose analogs able
to stimulate the phosphorylation of fructose without being substrate.
However, no such compound was found among analogs modified on C1 to C6.
Furthermore, the order of potency (glucose > mannose > 2-deoxyglucose) in the stimulation of fructose paralleled the capacity
of these compounds to act as substrates. The same was true for
glucosamine, which at low concentrations, was intermediate between
glucose and mannose both as a substrate and as a stimulator of fructose
phosphorylation. This, together with the lack of cooperativity in the
presence of poor nucleotide substrates and the hyperbolic aspect of
glucose binding to the enzyme, as estimated by protection from the
inactivation by
N-(N-bromoacetyl)-6-aminohexanoylglucosamine (17)
or by fluorescence spectroscopy (15), strongly argues against the
existence of an independent allosteric binding site.
Nature of the Slow Conformational Change: a
Hypothesis--
Because glucokinase is a monomer and because there is
no evidence for a second binding site for glucose, the explanation for the cooperativity for glucose must be kinetic. Because explanations such as a random order of addition of substrate (9) or the recycling of
an ADP-enzyme complex (32) do not apply to glucokinase (10), the best
models of cooperativity for glucokinase are those in which the enzyme
undergoes a slow conformational change between two states with
different affinities for glucose (11, 12). The stimulatory effect of
glucose on the phosphorylation of fructose (see Ref. 19 and this work),
mannose (19), and
2-deoxyglucose1
indicate that the state that has a higher affinity for glucose also has
a higher affinity for other substrates. As shown in this paper, it also
has a higher affinity for glucose analogs that act as inhibitors,
although not for other competitive inhibitors such as palmitoyl-CoA or
the regulatory protein of glucokinase.
The nature of this conformational change is still elusive as no crystal
structure of glucokinase is yet available, but the localization of
mutations that decrease cooperativity in the three-dimensional model of
glucokinase derived from the structure of yeast hexokinase B (30) may
be instructive in this respect. Mutations that decrease cooperativity
while considerably decreasing kcat values or the affinity for glucose are not informative in this respect, because their
effect is most likely due to the slowing down of the catalytic cycle,
similar to the effect of replacing ATP by poor phosphoryl donors in the
wild type enzyme. However, two mutations (N166R and Y214A) decrease
cooperativity while increasing the affinity for glucose and barely
changing Vmax values. Interestingly, they involve residues that are in the hinge region, at some distance from
the catalytic site, or at the interface between the two domains (22).
Because of the location of these residues, we postulate that, in
addition to the classical open and closed states common to all
hexokinases (33, 34), glucokinase would also exist in a
"super-open" state (Fig. 7). Because of the larger distance between
the glucose binding residues present in the larger and smaller domains,
this conformation would have a lower affinity for glucose and for its
analogs than the open conformation. For an as yet unknown reason, this
super-open form would only slowly equilibrate with the open
conformation. By binding to the super-open conformation, glucose would
facilitate partial closure of the catalytic cleft to the open state.
Binding of ATP-Mg, complete closure of the catalytic cleft, and the
phosphoryl transfer reaction would then ensue. Reopening of the
catalytic cleft (to the open state) would be rapidly followed by
extrusion of the products of the reaction, leaving the free enzyme in
its high affinity state. The enzyme would then be more prone to
catalyze the phosphorylation of any substrate (and to be inhibited by
substrate analogs). If no substrate comes in time, the enzyme would
"relax" to the super-open (low affinity) state. However, if
sufficient substrate is present, the enzyme "recruited" by glucose
to the open (high affinity) state can undergo several additional cycles
with another substrate such as fructose before relaxing to the
super-open state, explaining the more than stoichiometric stimulation
of fructose phosphorylation that we observed.
Functionally, this model is similar to the one proposed by Storer and
Cornish-Bowden (11) and differs from the slow transition model of
Cardenas et al. (12) in that catalysis can only occur with
the high affinity state. Testing of the model by mathematical simulations indicates that it can account for the sigmoidal behavior of
glucokinase with both glucose and fructose as substrates, for the
effect of mutations that decrease cooperativity, and for the stimulation of fructose phosphorylation by glucose. It can also account
for the differential behavior of N-acetylglucosamine and mannoheptulose on the kinetics of glucokinase if one admits that the
difference between the affinities of the two states is larger in the
case of mannoheptulose than in the case of
N-acetylglucosamine. This difference may be because of the
presence of a bulkier substituent on C2 than on the corresponding
carbon in mannoheptulose.