From the
Glucokinase is distinguished from yeast hexokinase and low
K
Glucokinase (ATP:D-hexose 6-phosphotransferase, EC
2.7.1.1) is expressed in pancreatic
The determinants of sugar specificity and cooperative behavior of
human
The pET3a C-HK (residues 456-920) expression
plasmid was prepared by digestion of pET3a HK-HK with NdeI and
NcoI, and the ends were filled in with Klenow and ligated
together. The pET3a N-HK (residues 1-455) expression plasmid was
prepared as follows. Polymerase chain reaction was used to amplify a
fragment of pET3a HK-HK using the T7 forward primer and a synthetic
primer, which contains a termination site right after the codon for
Ala-455 and a XbaI site. Both the polymerase chain reaction
fragment and pET3a vector were digested with XbaI. The pET3a
vector was further treated with phosphatase and ligated to the
XbaI-digested polymerase chain reaction fragment to construct
the pET3a N-HK. The authenticity of all plasmid constructs and fidelity
of the entire coding sequence of the cDNAs was determined by dideoxy
chain termination sequencing with modified T7 DNA polymerase.
Purifications of pET19b HK-HK and pET19b N-HK
were performed as follows. Cells harboring the expression plasmids were
induced and harvested as described for pET3a HK-HK. Cell pellets were
then lysed by freeze-thawing
Mutation of Asn-166 to Arg lowered the K
N166R
exhibited a 2-fold decrease in K
The results clearly show that the yields of
active hexokinase forms were better at 22 °C than 37 °C,
probably because the protein folds more readily at 22 °C. However,
N-HK was not active even though different strains and different
temperatures were tested (data not shown). Both HK-HK and C-HK were
induced (2-3 mg/liters) but not nearly to the same extent as
glucokinase (20 mg/liters). HK-HK and C-HK were partially purified by
15-30% ammonium sulfate fractionation and DEAE-A50 ion exchange
chromatography with a linear gradient of KCl (100-500
mM). The partially purified enzymes were free of bacterial
hexokinase. The kinetic properties of both HK-HK and C-HK were
characterized (). For HK-HK, the K
To confirm that the
N-HK is not active, it was necessary to undertake its purification.
However, this form was recalcitrant to purification (data not shown). A
His-tag system based on the T7 RNA polymerase expression system, in
which the HK cDNA is fused with a His-tag leader sequence, was used to
circumvent the problem. Comparison of the kinetic properties of HK-HK
with the His-tag sequence with the native HK-HK revealed no major
differences with respect to kinetic properties (). N-HK
with the His-tag sequence was induced and purified but had no
measurable activity. Western blots with a polyclonal antibody against
brain hexokinase confirmed the identity of the purified N-HK and HK-HK
(Fig. 3). The results provide definitive evidence that purified
N-HK has no enzymatic activity.
Mammalian low K
In contrast to the low
K
N-Acetylglucosamine is an effective competitive inhibitor
of glucokinase with respect to glucose. The K
ATPase activity is conserved in both yeast hexokinase and
glucokinase
(5, 19, 20, 21) . Mutation
of Ser-151 to Cys or Ala reduced the ATPase activity below detectable
levels, which suggests that the OH group of Ser-151 attacks the
In summary, this study has
provided clues to explain glucokinase's low affinity for glucose.
While mutation of most of the enzyme's putative glucose binding
residues dramatically increased the K
Glucokinase activity was assayed at 30 °C by measuring the
change in absorbance of NADPH at 340 nM as described under
``Experimental Procedures.'' Each data point represents the
average ± S.E. of triplicate independent curve fits. Each curve
was obtained from triplicate measurements at each of 9-12
different glucose concentrations. The range of glucose concentrations
for the wild-type and mutant enzymes used depended on the
K
Glucokinase activity was measured by monitoring the
decrease in absorbance at 340 nm using the lactate
dehydrogenase-pyruvate kinase-coupled assay as described under
``Experimental Procedures.'' The concentration of sugar
analogs used depended on the K
Glucokinase
activity was measured by using the glucose-6-phosphate dehydrogenase
coupling assay, with varying concentrations of
N-acetylglucosamine at different concentrations of glucose.
For Glu-6-P inhibition, glucokinase activity was measured by using the
pyruvate kinase-lactate dehydrogenase assay, with varying
concentrations of Glu-6-P at different concentrations of glucose.
V
Hexokinase activity was measured by using the
glucose-6-phosphate dehydrogenase coupling assay, with varying
concentrations of glucose with a range of 0.01-1 mM at 5
mM ATP or with varying concentrations of ATP with a range of
0.1-10 mM at 100 mM glucose. For Glu-6-P
inhibition assay, hexokinase activity was measured by using pyruvate
kinase-lactate dehydrogenase assay, with varying concentrations of
Glu-6-P at three different concentrations of ATP (0, 0.5, and 1
mM). V
Data for yeast HK are from Ref. 16. All the
other data are from Ref. 6 and this report. ND, not determined.
We thank Kristina Johnson for skillful typing of the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
mammalian hexokinases by its low
affinity for glucose and its cooperative behavior, even though glucose
binding residues and catalytic residues are highly conserved in all of
these forms of hexokinase. The roles of Ser-151 and Asn-166 as
determinants of hexose affinity and cooperative behavior of human
glucokinase have been evaluated by site-directed mutagenesis,
expression and purification of the wild-type and mutant enzymes, and
steady-state kinetic analysis. Mutation of Asn-166 to arginine
increased apparent affinity for both glucose and ATP by a factor of 3.
Mutation of Ser-151 to cysteine, alanine, or glycine lowered the
K
for glucose by factors of 2-, 26-, and
40-fold, respectively, decreased V
, abolished
cooperativity for glucose, and also decreased K
for mannose and fructose. The Ser-151 mutants had hexose
K
values similar to those of yeast
hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal
half of hexokinase I. However, the K
values for the competitive inhibitors,
N-acetylglucosamine and glucose-6-P, were unchanged,
suggesting that Ser-151 is not important for inhibitor binding.
Mutation of Ser-151 also increased the K
for ATP about 5-fold and abolished the enzyme's low ATPase
activity, which indicates it is essential for ATP hydrolysis. The
substrate-induced change in intrinsic fluorescence of S151A occurred at
a much lower glucose concentration than that for wild-type enzyme. The
results implicate a dual role for Ser-151 as a determinant of hexose
affinity and catalysis, exclusive of the glucose-induced conformational
change, and suggest that the low hexose affinity of glucokinase is
dependent on interaction of Ser-151 with other regions of the protein.
-cells and liver and is the
first rate-limiting reaction in glycolysis in these tissues
(1, 2, 3) . Glucokinase (hexokinase IV) is a
member of the hexokinase family of enzymes but is distinguished from
the other members of the family by its lower affinity for glucose
( K
= 6.0 mM compared with
20-170 µM for the low K
hexokinases), its molecular size (50 versus 100 kDa for
hexokinase), and its cooperative behavior with respect to glucose
(1, 2, 3, 4) . Recently, the sugar
specificity of human
-cell glucokinase has been studied by kinetic
analysis, fluorescence techniques, and molecular modeling
(5) .
The enzyme exhibits sigmoid behavior with glucose and 2-deoxyglucose as
substrates but not with mannose and fructose. A molecular model of
human
-cell glucokinase predicts that the carbonyl group of
Ser-151 interacts with the 2-OH of mannose and fructose while Glu-256
interacts with the 2-OH of the glucose, which suggests that Ser-151 may
be involved in the enzyme's cooperative behavior
(5) .
-cell glucokinase have also been studied by mutating several
putative active site residues
(6) , which are conserved in the
NH
- and COOH-terminal halves of mammalian hexokinase
(N-HK
(
)
and C-HK, respectively), glucokinase, and
yeast hexokinase
(7) . The conserved putative glucose binding
residues in human
-cell glucokinase are Ser-151, Asn-204, Asp-205,
Asn-231, Glu-256, and Glu-290
(8, 9, 10) .
Asp-205 was predicted, by analogy with yeast hexokinase, to be a base
catalyst, and consistent with that hypothesis, mutation of Asp-205 to
Ala decreased the k
of the enzyme by a factor of
at least 1000
(9) . Asn-204, Gly-256, and Glu-290 were predicted
from molecular modeling to interact with the 3-OH, 4-OH, 2-OH, and 1-OH
groups of glucose
(5, 10) . Mutation of these residues
resulted in enzymes with decreased values of k
and increased K
values for glucose,
mannose, and 2-deoxyglucose
(6) . These results support the
prediction of the model that these residues interact with hexoses
(5) . One conserved putative hexose binding residue of
glucokinase that has not yet been studied by site-directed mutagenesis
is Ser-151. In addition, based on the molecular model of glucokinase
(10) , Asn-166 has been postulated to constitute one of the
nonconserved residues whose corresponding residue in yeast hexokinase
may be important for ATP binding and the glucose-induced conformational
change
(11) . It was the object of this study to examine whether
Ser-151 and Asn-166 are involved in hexose binding and/or catalysis. It
is demonstrated here that point site mutations of Ser-151 but not
Asn-166 convert the high K
glucokinase to
a lower K
enzyme similar, in some of its
properties, to yeast hexokinase and the low K
mammalian hexokinases.
Materials
Q-Sepharose was from
Pharmacia Biotech Inc. Glu-6-P dehydrogenase, Lactate dehydrogenase,
and pyruvate kinase were from Boehringer Mannheim. Glucosamine,
N-acetylglucosamine, mannose, fructose, and all other sugars
were from Sigma. The full-length cDNA for human kidney hexokinase I was
a kind gift from Dr. Graeme Bell (Howard Hughes Medical Institute,
University of Chicago). Antibody to mouse brain hexokinase I was a kind
gift from Dr. John Wilson (Dept. of Biochemistry, Michigan State
University).
Construction of pET Expression Plasmids for Human
A 2.6-kilobase human pancreatic
-Cell Glucokinase
-cell glucokinase cDNA clone, ph-GK-20, was used to generate the
construct pEhgk-WT
(8) . An NdeI site was generated at
the 5`-end using the polymerase chain reaction with an oligonucleotide
that has a one-base pair mismatch (GGCTGGTGTGCATATGCTGGACGACAG). The
insert in the pET3a expression construct included the protein coding
region of the cDNA as well as the 3`-untranslated region.
Construction of pET3a HK-HK, N-HK, and C-HK
Expression Plasmids
The expression plasmid pET3a HK-HK was
constructed from an AlwNI- XbaI fragment of the human
kidney hexokinase cDNA and two pairs of complementary oligonucleotide
linkers. The linkers ( NdeI/ AlwNI, encoding the
NH-terminal amino acids and translation initiation site)
were phosphorylated, annealed, and ligated to the NdeI site of
the pET3a vector and the AlwNI site of the cDNA. The
3`-linkers ( XbaI/ EcoRI), encoding the COOH-terminal
phenylalanine and the termination site, were similarly treated and
ligated to the XbaI site of the cDNA and to the EcoRI
site of pET3a.
Construction of pET19b HK-HK, pET19b C-HK, and pET19b
N-HK
pET19b HK-HK was prepared by digesting the construct
pET3a HK and pET19b vector with NdeI and XbaI,
followed by ligation. Both pET19b C-HK and pET19b N-HK were prepared in
a similar manner.
Expression and Purification of HK-HK, C-HK, and
N-HK
The expression of HK-HK (intact HK), C-HK, and N-HK
were accomplished using the T7 RNA polymerase based system of Studier
and Moffat
(12) . Cells containing pET3a HK-HK and pET3a C-HK
were induced at 22 °C for 24 h and harvested, and cell pellets were
then lysed by freeze-thawing 3. The crude extracts containing
HK-HK and C-HK were partially purified by 30% ammonium sulfate
precipitation, and dialyzed against hexokinase buffer (5 mM
potassium P
, 1 mM EDTA, pH 8.0, and 10 mM
glucose). The enzyme fractions were loaded on to a 100-ml DEAE-A50 ion
exchange column and washed with hexokinase buffer containing 100
mM KCl. Hexokinase was eluted with a linear gradient of KCl
(100-500 mM). The pooled fractions were concentrated by
precipitation with 65% (NH
)
SO
,
dissolved in hexokinase buffer containing 100 mM KCl, and
purified to homogeneity by elution from a Sephadex G-100 superfine
column (1.5
90 cm). This purified preparation was free of
bacterial hexokinase.
3 except that a buffer containing
100 mM Tris, 100 mM KCl, pH 8.0, was used. The crude
extracts were passed through a DEAE-Sephadex column equilibrated in the
same buffer. The flow through was loaded onto a 10-ml Ni-Sepharose
column, and the column was washed with 100 ml of buffer containing 10
mM imidazole, 100 mM Tris, pH 8.0, and then eluted
with buffer containing 25 mM imidazole and 100 mM
Tris, pH 8.0. The hexokinase fractions were precipitated with 65%
ammonium sulfate, dissolved in 1 ml of hexokinase buffer, and purified
to homogeneity on a Sephadex G-100 superfine column (1.5
90 cm)
equilibrated with hexokinase buffer. The N-HK containing the His-tag
sequence was induced and purified to near homogeneity in a similar
manner. Western blots of both HK-HK and N-HK were performed using a
polyclonal antibody against rat brain hexokinase.
Site-directed Mutagenesis of Human
Mutagenesis of human -Cell
Glucokinase
-cell glucokinase was
carried out by using the method of Kunkel
(13) . A
NdeI- BamHI fragment of human pancreatic glucokinase
was subcloned into pBluescript. A single-stranded uracil-containing DNA
was prepared, annealed to a phosphorylated mutagenic oligonucleotide,
and transformed into XL-blue cells. Mutant clones were identified by
DNA sequencing. A NdeI- ClaI mutagenic fragment was
subcloned into the pET3a GK vector. The entire
NdeI- ClaI fragment was sequenced to confirm the
authentic sequence of the pET3a GK expression vector. The double mutant
(S151A and N166R) was prepared by subcloning the mutant fragment
( ClaI- SstI) of N166R into the S151A mutant expression
vector digested by ClaI and SstI. Oligonucleotide
primers were used to make mutations as follows with the mutated codon
in the lower case: GK (wild type),
5`-CCCCTGGGCTTCACCTTCTCCTTTCCTGTGAGG-3`; S151C,
5`-CCCCTGGGCTTCACCTTCtgcTTTCCTGTGAGG-3`; S151A,
5`-CCCCTGGGCTTCACCTTCgccTTTCCTGTGAGG-3`; S151G,
5`-CCCCTGGGCTTCACCTTCggcTTTCC-TGTGAGG-3`; and N166R,
5`-GAAGACATCGATAAGGGCATCCTTCTCagaTGGACCAAGGGC-3`.
Bacterial Expression and Purification of Native and
Mutant Forms of Human
Wild-type
glucokinase and mutant enzymes were expressed in Escherichia coli BL21(DE3)-plys S using the phage T7 RNA polymerase-based system
described by Studier and Moffat
(12) , except that a lower
induction temperature was used
(9) . Similar yields of about 20
mg/liter were obtained for all of the mutants and for the wild-type
enzyme. Cell extracts containing glucokinase were freeze-thawed three
times to break the cells, and 100 µg/ml DNase A and 1 mg/ml
lysozyme were added to the cell extract at 4 °C for 1 h.
Glucokinase was first partially purified by precipitation with
40-65% (NH-Cell Glucokinase
)
SO
. The pellet was
dissolved in glucokinase buffer (50 mM potassium
P
, pH 8.0, 1 mM EDTA, 1 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride),
dialyzed against the same buffer, and then loaded on to a 100-ml
Q-Sepharose column. The column was washed with 2 liters of glucokinase
buffer containing 150 mM KCl, and the enzyme was eluted with a
linear gradient of 150-450 mM KCl. The fractions
containing enzyme activity were pooled, precipitated with 70%
(NH
)
SO
, and dissolved in 1 ml of
glucokinase buffer. Glucokinase was loaded on to a 100-ml G-100
Sephadex superfine column equilibrated in glucokinase buffer. The
wild-type and mutant enzymes were eluted from the column with an
apparent molecular mass of 50 kDa. Native human
-cell glucokinase
has a specific activity of about 80-120 units/mg, which is
similar to that reported for the purified rat liver enzyme
(14) . The various Ser-151 mutants were also purified to
homogeneity by the above procedure.
Enzyme Assay and Kinetic
Analysis
Glucokinase activity was assayed at 30 °C by
measuring the increase in absorbance of NADPH at 340 nm in a coupled
enzyme system, which employed Glu-6-P dehydrogenase as previously
described
(5, 9) . The Glu-6-P dehydrogenase assay was
also used to determine the Kfor
N-acetylglucosamine inhibition. When sugar specificity was
studied, glucokinase activity was measured by monitoring the decrease
in absorbance at 340 nm in an assay mixture containing 100 mM
Tris-HCl (pH 7.5), 100 mM KCl, 20 mM
MgCl
, 0.6 mM phosphoenolpyruvate, 0.6 mM
NADH, 1 mM ATP, 1.76 units/ml lactate dehydrogenase, and 1.4
units/ml pyruvate kinase. The K
for
Glu-6-P, with respect to glucose, was determined using the pyruvate
kinase-lactate dehydrogenase coupling assay. The V
and K
for substrates were obtained
by the nonlinear least squares fitting program to fit a
Michaelis-Menten equation or V = V
S ( K
+ S) for the Hill
coefficient using Sigma Plot.
Modeling of Human
A model of human -Cell Glucokinase
Structure
-cell glucokinase was
generated by analogy to the known crystal structure of the related
yeast hexokinase
(10, 11) followed by molecular
mechanics minimization as described by Xu et al. (5) .
The model and crystal structures were examined using the program FRODO
(35) run on an Evans and Sutherland ESV 10 computer graphics
workstation
(5, 10) .
Determination of Circular Dichroism
Spectra
All far UV spectra were collected on a Jasco 500 A
in a 0.01-cm cell at 20 °C. Scans were collected at 1 nm/min with a
time constant of 64 s, and all data are the average of three blank
corrected samples. Samples were dialyzed against 50 mM
potassium P, pH 7.5, 1 mM EDTA, and 1 mM
dithiothreitol. Samples were centrifuged at 14,000 rpm for 10 min to
remove any precipitated protein, and the A
was
measured to scale the CD data to the same concentration. The CD data
presented corresponds to samples with an A
of
1.0 in a 1-cm cell.
Determination of Intrinsic Fluorescence of
Experiments were performed in a buffer
containing 50 mM potassium P-Cell
Glucokinase
, pH 7.5, 1
mM MgCl
, 1 mM EDTA, 1 mM
dithiothreitol, and 5% glycerol with a multi-frequency phase
fluorometer (ISS Inc.). The excitation wavelengths were 280 nm in a 10
10-mm
size cuvette
(6, 15) . Glucose
was removed from glucokinase prior to the experiment by a dialysis
overnight against a buffer containing 50 mM potassium
P
, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol. The experiment was initiated by the addition of a final
concentration of 1 or 100 mM hexose. The fluorescence spectra
were recorded after addition of sugar and then analyzed with an IBM-PC
microcomputer interfaced with the fluorometer to extract maximum
fluorescence intensity of spectra at 312 nm. The relative fluorescence
enhancement was obtained by dividing fluorescence enhancement by the
initial intrinsic fluorescence.
Assay of ATPase Activity of
Glucokinase
Reaction mixtures for following the ATPase
activity of glucokinase contained 0-20 mM
Mg-[-
P]ATP (3
10
cpm/umol), 5 mM excess MgCl
, 50 mM
Pipes, pH 7.0, and 20 µg of glucokinase in 200 µl. Inorganic
phosphate release was performed by the method of Viola et al. (16) as previously described
(5) .
Expression and Purification of Wild-type and Mutant
Glucokinases
Native and mutant human -cell
glucokinases were expressed in E. coli using the pET
expression system essentially as previously described
(5, 8, 9) . Glucokinase was purified 20-fold
from extracts of E. coli by
(NH
)
SO
precipitation
(45-65%), Q-Sepharose chromatography, and gel filtration on a
Sephadex G-100 superfine column as described under ``Experimental
Procedures''
(20) . The Q-Sepharose column is the most
important step for removing contaminating bacterial hexokinase, which
was eluted with the 150 mM KCl, potassium P
, pH
8.0, wash. Wild-type glucokinase and all five Ser-151 and Arg-166
mutants were purified to homogeneity by the criteria of SDS-PAGE (data
not shown).
Circular Dichroism of the Mutants and Wild-type
Enzyme
To ascertain whether the point site mutations caused
large changes in secondary structure, the circular dichroism spectra of
the mutants were compared with wild-type enzyme. There were no
significant changes in the circular dichroism spectra of the Ser-151
mutants (Fig. 1), which indicates that there was no gross change
in their secondary structure. There was also no change in the circular
dichroism of the Asn-166 mutants (data not shown).
Figure 1:
CD
spectra of wild-type glucokinase and various Ser-151 mutants. The
circular dichroism data were blank corrected and scaled for
concentration.
Effect of Mutation of Ser-151 and Asn-166 on
Glucokinase Phosphorylation of Glucose
Two distinctive
features of mammalian glucokinase are its high
Kfor glucose and cooperative behavior
with respect to glucose
(1, 2, 3, 4) .
The effect of various Ser-151 mutations is shown in .
Mutation of Ser-151 to cysteine decreased the K
for glucose to one-third, while k
decreased to about one-tenth the wild-type value. Mutation of
Ser-151 to Ala decreased the K
for
glucose by a factor of nearly 20, while k
decreased to one-tenth the wild-type value. The S151G mutant had
a K
about 1/40th the wild-type value,
while k
decreased by 2 orders of magnitude. The
K
values for ATP were increased about
5-fold for all Ser-151 mutants. The sigmoid behavior of glucokinase
with regard to glucose was abolished for the S151A and S151G mutants.
values for glucose and for ATP to one-third the wild-type enzyme
value, while V
and the Hill coefficient were not
affected. Therefore, the effect of mutating both Ser-151 and Asn-166
was tested. The double mutant, N166R/S151A, exhibited a
K
for glucose about 60% lower than that
for the S151A mutant, but the V
was also
decreased by a factor of 3. Like the S151A mutant, the double mutant
had a Hill coefficient of 1.0 and a higher K
for ATP.
Effect of Mutation of Ser-151 and Asn-166 on Sugar
Specificity of Glucokinase
The sugar specificity of human
-cell glucokinase has been studied
(5) . Mannose,
2-deoxyglucose, and fructose are substrates of the enzyme, and it was
of interest to determine whether the Ser-151 mutants also had enhanced
affinities for these substrates. As shown in , for the
S151C mutant, the K
values for mannose
and fructose were similar to that for the wild-type enzyme, but the
k
values relative to glucose were decreased. The
S151A mutant exhibited decreased K
values
for mannose, 2-deoxyglucose, and fructose by factors of about 20, 10,
and 4, respectively, compared to the wild-type enzyme. The
V
values decreased by factors of 20 for mannose
and 2-deoxyglucose and by about 5-fold for fructose compared to
wild-type enzyme. The Ser-151 mutants had similar catalytic
efficiencies for glucose and mannose, except for S151C, which
phosphorylated mannose less efficiently than glucose. S151A was similar
to the wild-type enzyme in catalytic efficiency for fructose, but the
Cys and Ala mutants were at least 1 order of magnitude less efficient
at phosphorylating fructose compared to the wild-type enzyme.
for
mannose compared to the wild-type enzyme, resulting in nearly 2-fold
greater catalytic efficiency. For fructose, this mutant showed an
increase in V
and decreased
K
, resulting in nearly 10-fold greater
catalytic efficiency compared to the wild-type enzyme. The double
mutant had greatly enhanced affinities for mannose and fructose.
Effect of Ser-151 Mutations on Glucose-induced
Changes in Intrinsic Fluorescence
Rat liver glucokinase
exhibits a glucose-induced change in intrinsic fluorescence that has
been postulated to reflect the presence of two different conformations
of the enzyme
(15, 17) . In the crystal structures,
yeast hexokinase is observed in an open conformation in the presence of
a glucose analog inhibitor and in a closed conformation in the complex
with glucose
(18) . Human -cell glucokinase has been
predicted to undergo a similar conformational change on the binding of
glucose
(5, 6, 10, 18) . For the
wild-type enzyme, 100 mM glucose caused a 31.1% increase in
intrinsic fluorescence, while 1 mM glucose had no significant
effect. The S151C mutant exhibited a small but significant increase
(4.2%) in intrinsic fluorescence at 1 mM glucose and the same
change at 100 mM glucose as seen with the wild-type enzyme.
However, the S151A mutant exhibited a 41.5% change in intrinsic
fluorescence at 1 mM and a 54.8% increase at 100 mM
glucose. The results show that in the case of the S151A mutant, the
glucose dependence of changes in intrinsic fluorescence parallels the
changes in hexose affinity. The N166R mutant exhibited a reduced
glucose-induced change in intrinsic fluorescence (4% at 1 mM
glucose and 10% at 100 mM glucose).
Effect of Mutations on Inhibition of Glucokinase by
N-Acetylglucosamine and Glu-6-P
I shows that
the Kvalues for the competitive
inhibitor N-acetylglucosamine
(5, 6, 18) for all of the Ser-151 mutants were similar to that of
wild-type enzyme, even though the S151A and S151G mutants exhibited
dramatically decreased K
values for
glucose. The K
values for Glu-6-P of all
Ser-151 mutants were also similar to that for the wild-type enzyme,
with small decreases for S151C and S151A (I). The results
demonstrate that mutation of Ser-151 has little effect on the
enzyme's inhibition by Glu-6-P and N-acetylglucosamine.
The N166R mutant and the double mutant showed increased affinity for
N-acetylglucosamine by about 2-3-fold but no change in
K
for Glu-6-P.
Effects of the Ser-151 Mutations on ATPase Activity
of Glucokinase
Yeast hexokinase exhibits low ATPase
activity
(19, 20, 21) , which is inhibited by
phosphorylation of Ser-158, a side reaction product of ATP hydrolysis
(20, 21) . Glucokinase also has a low ATPase activity,
and Ser-151 corresponds to Ser-158 of yeast hexokinase
(10) .
Mutation of Ser-151 to Cys or Ala abolished the ATPase activity as
shown in Fig. 2. For comparison, the N204Q mutant showed easily
detectable ATPase activity, even though Vdecreased by 10-fold compared to the wild-type enzyme
(6) . The specific activities ( V
) of the
kinase reaction were similar for the N204Q (6 units/mg), S151C (6
units/mg), and S151A (6 units/mg) mutants. No ATPase activity was
measured with the Ser-151 mutants, even when three times more mutant
enzyme protein than wild-type enzyme was used. These results suggest
that Ser-151 is directly involved in the hydrolysis of ATP.
Figure 2:
ATPase activity of human -cell
glucokinase and various mutants. Glucokinase ATPase activity was
measured by following the production of
P from
[
-
P]ATP as described under
``Experimental Procedures.'' Eight different ATP
concentrations in the range of 0-20 mM were
used.
Kinetic Analysis of HK-HK, C-HK, and
N-HK
Mutation of the active site residue Ser-151 converts
the high Kglucokinase to a low
K
enzyme. To compare the glucokinase
Ser-151 mutant forms with low K
hexokinases of the same molecular size, the cDNAs corresponding
to the NH
- and COOH-terminal halves of hexokinase I were
engineered into expression vectors. The BL 21(DE3)pLysS strains
harboring pET3a HK-HK, C-HK, and N-HK were induced at 37 and 22 °C
by isopropyl-1-thio-
-D-galactopyranoside. The cells
harboring the pET3a vector were used as a control. The yield of active
soluble protein at 37 °C was low (0.3 mg/liter). HK-HK was induced
rapidly at this temperature and reached a plateau at 5 h and decreased
afterward. C-HK was induced more quickly and reached a plateau at 10 h.
In contrast, N-HK was induced very well as judged by
[
S]Met incorporation (data not shown), but
hexokinase activity was insignificant, suggesting that N-HK is not
active. In contrast to the low yield of active soluble enzyme at 37
°C, the yield of soluble, active C-HK was 10-fold higher at 22
°C (3 mg/liter). HK-HK was also induced to a higher level at this
temperature and reached a plateau of 2.5 mg/liter at 20 h. As was the
case at 37 °C, N-HK was induced very well at 22 °C (data not
shown), but again the hexokinase activity was the same as that of the
pET3a vector alone.
for glucose was 28.2 µM, the
K
for ATP was 0.47 mM, and the
K
for Glu-6-P was 70 µM.
Interestingly, C-HK retained essentially the same kinetic constants as
the intact hexokinase (HK-HK) (). The results suggest that
the Glu-6-P inhibition site and catalytic site are located in the C-HK
domain, consistent with the notion that Glu-6-P binds at the catalytic
site and/or its binding site overlaps with it.
Figure 3:
A,
SDS-PAGE of purified His-tag-HK-HK and His-tag-N-HK. About 10 µg of
purified His-tag-HK-HK and His-tag-N-HK were subjected to SDS-PAGE.
Lanes 1-3 are molecular marker, HK-HK, and
N-HK, respectively. B, Western blot of purified His-tag-HK-HK
and His-tag-N-HK. About 10 µg of purified His-tag-HK-HK and
His-tag-N-HK were subjected to SDS-PAGE and transferred to
nitrocellulose membrane. The membrane was blotted with nonfat milk and
probed with polyclonal antibody against mouse brain hexokinase I.
Lanes 4 and 5 are HK-HK and N-HK,
respectively.
hexokinases are
not expressed in high yield in bacterial expression systems
(28, 29, 30) in contrast to the lower molecular
weight glucokinases
(6, 8, 9) , even though
NH
- and COOH-terminal halves of hexokinase share extensive
similarity with glucokinase
(22, 23, 24, 25, 26) .
Expression and characterization of the NH
- and
COOH-terminal halves of hexokinase revealed that C-HK retained the same
catalytic activity, with a high affinity for glucose and a marked
inhibition by Glu-6-P, as intact native hexokinase I
(27, 28, 29) . This is the first instance where
the NH
- and COOH-terminal halves of low
K
hexokinase have been purified. The
NH
-terminal form had no activity in its native form (pET3a
vector) or as a fusion protein (pET19 vector), while the intact
hexokinase was active in both instances. The results strongly suggest
that the NH
-terminal half of hexokinase I is not
responsible for the enzyme's catalytic activity or sensitivity to
Glu-6-P inhibition. It is possible the NH
-terminal half may
mediate hexokinase I interaction with the mitochondria or other
subcellular structures
(31, 32) . It is not known
whether the NH
-terminal halves of hexokinase II or III are
also inactive. Hexokinase II has been postulated to have arisen as a
result of gene duplication of a ``glucokinase''-like gene
(23, 33) . If this is true, the gene duplication and
tandem ligation of the two hexokinase halves per se may be responsible for the low affinity for glucose, or
alternatively, subsequent mutations in the NH
- or
COOH-terminal halves may have resulted in an increase in glucose
affinity. The former possibility seems unlikely in view of the findings
with hexokinase I reported here. This question can be approached by
characterization of the NH
- and COOH-terminal halves of
hexokinase II, and such work is in progress.
hexokinases, glucokinase has a low
affinity for glucose ( K
= 6
mM) and does not exhibit product inhibition by physiological
concentrations of Glu-6-P ( K
= 12
mM)
(1, 2, 3, 4) (). Yeast hexokinase shares 33% identity with
glucokinase but exhibits a high affinity for glucose
( K
= 100 µM) and lack
of inhibition by Glu-6-P ( K
= 20
mM)
(16) , even though all the putative glucose-binding
residues are conserved in C-HK, N-HK, glucokinase, and yeast hexokinase
(22, 23, 24) . Recently, we have shown that
mutation of the active site residues, Asn-204, Glu-256, or Glu-290 to
Ala in the human
-cell glucokinase results in an increased
K
for glucose and a decreased
k
with loss of sigmoid behavior with glucose as
substrate. The cooperative behavior of glucokinase may thus be mediated
by interactions of other nonconserved regions of the protein with the
highly conserved active site glucose-binding residues
(6) .
Analysis of the crystal structure of yeast hexokinase led to the
selection of Asn-166 as one such nonconserved residue. Its location,
and that of Ser-151, in the glucokinase structure is shown in
Fig. 4
, which depicts a stereo view of the open form of
glucokinase based on the crystal structure of yeast hexokinase
(5, 10, 11) . Both residues are located in the
active site cleft in the smaller of the two domains. In the open
conformation of yeast hexokinase, the residue corresponding to
glucokinase 166 is Arg-173, and Arg-173 was predicted to form an
interdomain hydrogen bond interaction with Gln-298
(11) . The
Arg-173 to Gln-298 interaction spans the sugar-binding cleft and
possibly may be involved in the conformational change induced by
substrate binding. In addition, Arg-173 provides a positively charged
residue near the expected ATP-binding site. Mutation of Asn-166 to Arg
in glucokinase would restore this positive charge, which would be
predicted to promote the binding of ATP and therefore the
phosphorylation of sugar. These predictions were born out by kinetic
analysis of the N166R mutant, which revealed an enhanced affinity for
both ATP and glucose. However, this single point site mutation did not
affect cooperativity or convert the high K
enzyme to a low K
hexokinase-like
form, although its apparent affinity for mannose and fructose was also
increased.
Figure 4:
Stereo view of the open conformation of
human glucokinase. The mutated residues Ser-151 and Asn-166 are located
close to the bound glucose in the model structure of human glucokinase.
The carbon backbone of the glucokinase model is shown
( continuous line) with the side chain atoms of
Ser-151, Asn-166, and glucose ( ball and stick representation).
This report strongly suggests that Ser-151 plays a
critical role in determining the hexose affinity and cooperativity, as
well as catalysis, of the enzyme. The residue equivalent to Ser-151 in
yeast hexokinase is Ser-158, which is phosphorylated during the ATPase
reaction. Also, molecular modeling of glucokinase predicted that the
carbonyl oxygen of Ser-151 would form a hydrogen bond interaction with
the 2-hydroxyl group of mannose
(5) . This analysis suggested
that 1) Ser-151 was located near the phosphate of ATP and 2) Ser-151
was implicated in the cooperative effect of glucose
(5) . When
the crystal structures of yeast hexokinase in the open and closed
conformations are compared, differences are seen in the position and
interactions of Ser-158. In the open conformation of yeast hexokinase,
the hydroxyl side chain of Ser-158 was within hydrogen bonding distance
of the amino acid side chains of Asp-86 and Lys-176. However, after the
conformational change to the closed form, Ser-158 moved closer to
glucose so that its hydroxyl group still interacted with Asp-86 but no
longer interacted with Lys-176, and the carbonyl oxygen of Ser-158
formed a hydrogen bond interaction with the 3-hydroxyl group of
glucose. Apparently, only after the conformation change does Ser-158
become a glucose-binding residue. We propose that a similar
conformational change occurs in glucokinase. This suggests an
explanation for the lower Kaccompanied
by a lower reaction rate for the Ser-151 mutants. In each of the
mutations of Ser-151 to Cys, Ala, and Gly, the hydroxyl side chain of
Ser and any interactions with the neighboring Asp-78 and Lys-169 have
been eliminated. This may facilitate the conformation change and
movement of residue 151 to the position in which the carbonyl oxygen
can interact with the 3-hydroxyl group of glucose (Fig. 5), thus
increasing the apparent affinity for glucose. Alternatively, the
non-productive binding of hexoses or ATP may be reduced by eliminating
potential interactions with residue 151. The glucose-induced change in
conformation of residue 151 may be more effective when the Ser hydroxyl
group and its interactions with as yet undefined neighboring residues
are eliminated.
Figure 5:
Top panel, schematic
representation of the interactions of Ser-151 in the open conformation
of human glucokinase as predicted by analogy with the crystal structure
of yeast hexokinase B. Potential hydrogen bond interactions are
indicated by dashed lines. Ser-151 interacts with the
side chains of Asp-78 and Lys-169. Lower panel,
schematic representation of the interactions of Ser-151 in the closed
conformation of human glucokinase as predicted by analogy with the
crystal structure of yeast hexokinase A with glucose. Potential
hydrogen bond interactions are indicated by dashed lines. Ser-151 interacts with the side chain of Asp-78
and the 3-OH of glucose.
However, the loss of hydrogen bond interactions
among the residues forming the glucose binding site is expected to
destabilize the enzyme structure and result in a reduced reaction rate.
For example, mutation of Asn-204, Glu-256, and Glu-290 to Ala in the
human -cell glucokinase resulted in increased
K
values for glucose, decreased
k
values, and diminished conformational changes
upon addition of 100 mM glucose, as measured by the change in
intrinsic fluorescence
(6) . In the case of Ser-151 mutations,
the greatest decrease in catalysis was observed for the S151G mutant,
which is probably less stable due to the flexible glycine. S151A is the
most effective mutation, since it apparently retains some structural
stability due to the short alanine side chain and undergoes the
glucose-induced conformational change at low glucose concentration, as
shown by fluorescence changes. The residue in hexokinase I
corresponding to Ser-151 in glucokinase has been mutated with loss of
enzyme activity
(33, 34) consistent with the results
reported here, but the mutated enzyme was not purified nor was its
kinetic properties characterized in detail.
values for N-acetylglucosamine were unchanged for the
various Ser-151 mutants even though K
for
glucose was dramatically decreased to values near that for low
K
hexokinase and yeast hexokinase. These
results suggest that N-acetylglucosamine is not contacted by
Ser-151. Consistent with this hypothesis, Xu et al. (5) modeled N-acetylglucosamine binding to the open
conformation of the enzyme and predicted that Ser-151 was not involved
in binding the inhibitor. This report also confirms previous studies
that the catalytic site of mammalian low K
hexokinase with high affinity for glucose and Glu-6-P is in the
COOH-terminal half of hexokinase, while N-HK is not active
(27, 28, 29) . Although the S151A and S151G
mutants have similar affinity for glucose as do C-HK and yeast
hexokinase (), mutation of Ser-151 did not generate a
sensitive Glu-6-P inhibitory site in glucokinase, suggesting that the
Ser-151 mutants resemble yeast HK more than C-HK ().
-phosphate of ATP. In the kinase reaction, the carboxylate group
of Asp-205 is involved in transferring the
-phosphate of ATP to
the 6-OH group of glucose
(9) . The Ser-151 mutations also
resulted in about a 5-fold increase in the K
for ATP and reduced the V
. The results
reported here suggest that Ser-151 is physically close to the base
catalyst, Asp-205. In the yeast hexokinase closed form crystal
structure, the side chains of the equivalent Ser-158 is 4.4 A from the
side chain of the catalytic Asp-211.
for
glucose
(6) , mutation of Ser-151 decreased the
K
for glucose into the range found in
yeast hexokinase and low K
hexokinases
(), eliminating cooperative behavior and converting the
high K
glucokinase to a low
K
enzyme but with a compromised catalytic
rate. This implies that it might not be possible to convert the high
K
to a low K
enzyme by a single point mutation, with no change in
k
. This view is supported by results of the
double mutation, N166R/S151A. However, as judged by changes in
intrinsic fluorescence of S151A, the glucose-induced conformational
change occurs at an even lower glucose concentration than for the
wild-type enzyme, suggesting that the lower k
is
not due to decreased ability of the enzyme to undergo the
glucose-induced conformational change but rather is due to distortions
in the active site or related protein regions that prevent the
efficient transfer of the
-phosphate of ATP to the 6-OH group.
However, it also leaves open the possibility that a low
K
``glucokinase'' with no
change in turnover number can be generated by site-directed mutagenesis
of Ser-151 and other nonconserved residues in glucokinase. Such work is
in progress.
Table: Effect of mutation of Ser-151 on -cell glucokinase catalysis and affinity for glucose
of the enzyme. For the wild-type
enzyme, glucose concentrations in the range of 0.1-100 mM were used. For the S151G mutant, glucose concentrations in the
range of 0.05-1 mM were used. V
,
K
, and Hill coefficient were determined
by the non-linear least squares routine using Sigma plot.
Table: Effect of mutation of
Ser-151 and Asn-166 on the sugar specificity of -cell
glucokinase
of the
enzyme and was between 0.05 and 600 mM.
V
, K
were
determined by non-linear least squares routines using Sigma plot. ND
designates not determined.
Table: Effect of
mutation of Ser-151 on competitive inhibition of -cell
glucokinase by N-acetylglucosamine and Glu-6-P
and apparent K
were determined by using non-linear least squares methods to fit
the Michaelis-Menten equation using Sigma plot.
K
was obtained by Dixon plot (l/v
versus I).
K
/ K
represents the ratio of the K
for
inhibition over the K
for glucose. The
data represent the averages of three separate determinations.
Table: Kinetic properties of hexokinase
and apparent
K
were determined by using non-linear
least squares methods to fit the Michaelis-Menten equation using Sigma
plot. K
was obtained by Dixon plot (l/v
versus I). ND designates not determined. - designates no
inhibition for the bacterial form. The data represent averages of three
separate determinations of each kinetic parameter.
Table: Comparison of N-HK, C-HK, yeast hexokinase,
and glucokinase
-terminal half of human
kidney hexokinase I; C-HK, the COOH-terminal half of human kidney
hexokinase I; HK-HK, human kidney hexokinase I; Pipes,
1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; GK, glucokinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.