From the
The truncated gene of hexokinase, mini-hexokinase, starting with
methionine 455 and ending at the C terminus was expressed in
Escherichia coli. Mini-hexokinase lost its ability to
ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the
presence of phosphate (P
Brain hexokinase, hexokinase I (ATP:D-hexose
6-phosphotransferase, EC 2.7.1.1), is thought to be the pacemaker of
glycolysis in brain tissue
(1) and the erythrocyte
(2) .
The cDNAs for hexokinases from a variety of tissues, including yeast
(3, 4) , human kidney
(5) , rat brain
(6) , rat skeletal muscle
(7) , rat liver
(8, 9) , and mouse hepatoma
(10) have been
cloned, and their complete amino acid sequences have been deduced from
the cloned cDNAs. The C-terminal domains of mammalian hexokinases and
their N-terminal domains have overall similarities to each other and to
yeast hexokinase, probably as a result of gene duplication and fusion
(11, 12, 13, 14, 15) . The
crystal structure of yeast hexokinase is available
(16, 17) , and the putative glucose-binding domain has
been suggested from these investigations
(8, 18) .
The involvement of a cysteinyl residue either in or near the active
site of brain hexokinase
(19, 20, 21, 22) has been suggested. Swarup and Kenkare
(23) were
the first investigators to use
N-(bromoacetyl)-D-glucosamine to covalently modify
brain hexokinase. They demonstrated that two sulfhydryl residues are
involved in covalent modification with concomitant enzyme inactivation;
however, the enzyme could be protected against inactivation by either
ATP or glucose. Recently, Schirch and Wilson
(24) used this
same protocol with rat brain hexokinase and obtained results similar to
those reported by Swarup and Kenkare
(23) and concluded that
there was a single critical sulfhydryl based on the observation that
modification of that sulfhydryl totally inactivated the enzyme. Later,
in a subsequent study, they isolated and partially sequenced two
peptides that are modified by
N-(bromoacetyl)-D-glucosamine and that are protected
against modification by either glucose or N-acetylglucosamine
(25) . By locating these two peptides within the tertiary
structure of yeast hexokinase, based on the structure proposed by
Steitz and co-workers
(16, 17) , they found that these
peptides are close to the binding site for glucose. Comparing the amino
acid sequences between yeast hexokinase and the brain hexokinase
peptides, they found no cysteine residues corresponding to the peptides
in question. Based on the proximity of the peptides to the
glucose-binding site and the analysis of sequence homology between
yeast hexokinase and those peptides, they questioned the existence of a
single critical sulfhydryl. To solve this problem directly, we mutated
Cys
In 1989
White and Wilson
(27) reported studies on the selective tryptic
cleavage of rat hexokinase I into discrete N- and C-terminal halves.
They found that the active site of the enzyme resides in the C-terminal
half of hexokinase I, and they suggested that the allosteric site for
glucose-6-P is contained in the N-terminal segment of brain hexokinase.
Their kinetic results suggest that the C-terminal portion of brain
hexokinase, mini-hexokinase, is virtually identical to the intact
enzyme. Recently, Magnani et al.(28) obtained
mini-hexokinase from human tissue by recombinant technology and, like
White and Wilson
(27) , found that the truncated enzyme is very
similar in kinetic properties to human hexokinase I.
In the present
study, we report the functional expression of human brain
mini-hexokinase in Escherichia coli and its purification to
homogeneity. Site-directed mutagenesis was performed to obtain two
mutants of mini-hexokinase, C606S and C628S, in order to evaluate the
roles of these two cysteinyl residues in the hexokinase reaction.
Although our laboratory has cloned and expressed full-length human
brain hexokinase in E. coli, we chose to investigate
mini-hexokinase to simplify interpretation of the site-specific
mutagenesis data and, in addition, to study the effects of inorganic
phosphate (P
Because the crystal structure of yeast hexokinase did not contain
ATP, the location of this substrate has not been defined. On the other
hand, in actin and hsp70 heat shock protein there is a binding site for
ATP, and it has been suggested
(29, 30, 31) that a similar ATP-binding domain may exist on hexokinase.
To test this hypothesis, we used the structures of hsp70 and actin as a
model in an attempt to locate the nucleotide-binding site on human
brain hexokinase by using the technique of site-directed mutagenesis.
The finding by White and Wilson
(27) that brain
hexokinase could be cleaved into two roughly equal segments of which
the C-terminal half is very similar, if not identical, in properties to
the native enzyme is extremely interesting. This observation is fully
consistent with the suggestion that hexokinase I arose through gene
duplication and fusion
(11, 12, 13, 14, 15) . Our
studies with recombinant mini-hexokinase differ from those of Magnani
et al.(28) only in the system used to express the
enzyme. When we tested glucose-6-P-inhibited mini-hexokinase with
levels of P
In 1962 we found that glucose-6-P is a normal
competitive product inhibitor of ATP in the brain hexokinase reaction
(40) . This finding was clearly at variance with data from the
laboratory of Crane and Sols
(41) . It is now widely accepted
that glucose-6-P is indeed a competitive inhibitor of ATP
(28, 40, 42, 43) . Although we suggested
that glucose-6-P is a normal product inhibitor of brain hexokinase,
others, notably Lazo et al.(44) and Wilson
(45) , have maintained that glucose-6-P binds at an allosteric
site on hexokinase I. Reports from our laboratory, based primarily on
kinetic studies
(40) , as well as NMR experiments from other
laboratories
(46, 47) are clearly at variance with the
notion that a topologically distinct site for glucose-6-P exists on
hexokinase I. In addition, initial rate studies from Sols laboratory
(48) as well as our laboratory
(49) on the back reaction
clearly eliminate the existence of a high affinity glucose-6-P
inhibitory allosteric site. The finding by White and Wilson
(27) that mini-hexokinase is as susceptible to glucose-6-P
inhibition, both quantitatively and qualitatively, as the native brain
enzyme is fully consistent with the proposition that an allosteric site
for the sugar phosphate product does not exist. What is clear from the
present report is that the allosteric site for P
In 1992, Bork et al. and others
(29, 30, 31) indicated that the heat shock
protein hsp70, actin, and yeast hexokinase have similar three-
dimensional structures in their ATP-binding sites. The ATP-binding site
consists of five motifs: PHOSPHATE 1, CONNECT 1, PHOSPHATE 2,
ADENOSINE, and CONNECT 2. The ATP-binding motif is distinctly different
from the single phosphate-binding loop in, for example, adenylate
kinase, rec A protein, or ras p21
(50) . Amino acid residues in
the ATP-binding site are conserved although no overall sequence
similarity among these three proteins is found. In particular, the
PHOSPHATE 1 motif, which interacts with the
A number of studies
have shown that there is an essential thiol group at the active site of
hexokinase I
(22, 23, 24) . Schirch and Wilson
(25) isolated three peptides containing modified sulfhydryl
groups after trypsin digestion of the enzyme. They found that labeling
of two peptides, one containing Cys
). We suggest that the P
site either resides in the N-terminal half of hexokinase I or
requires the N-terminal portion of the enzyme. Site-directed
mutagenesis was performed to obtain two mutants of mini-hexokinase:
C606S and C628S. Both are thought to be associated with the active site
of hexokinase I. These mutants exhibited a 3-fold increase in
K
for glucose but no change in either the
K
for ATP or the
k
. The circular dichroism (CD) spectra showed no
differences among the wild-type or mutant enzymes. These results
suggest that Cys
and Cys
are not involved
in glucose binding directly. The putative ATP-binding site of
full-length human brain hexokinase may involve Arg
and
Gly
, and these residues were mutated to Ile. For the
mutant R539I, the k
value decreased 114-fold
relative to wild-type hexokinase, whereas the K
values for ATP and glucose changed only slightly. No change was
observed in the K
value for
1,5-anhydroglucitol 6-phosphate. CD spectra showed only a slight change
in secondary structure. For the mutant G679I, overexpressed hexokinase
is insoluble. We suggest that Arg
is important for
catalysis because it stablizes the transition state product
ADP-hexokinase. Gly
is probably important for proper
folding of the protein.
and Cys
, which are located in those two
peptides. Working with adenylosuccinate synthetase
(26) , we
found a thiol residue to be essential when covalently modified with
5,5`-dithiobis(2-nitrobenzoic acid); however, site-specific mutagenesis
of the specific cysteinyl residue in question revealed that the
covalent modification caused inactivation through a steric effect,
i.e. the enzyme could not fold into its native conformation
after modification with 5,5`-dithiobis(2-nitrobenzoic acid).
) on glucose-6-P-inhibited mini-hexokinase.
Materials
The pET-11d plasmid was obtained from
Novagen. E. coli strains BL21(DE3) and TG-1 cells were
obtained from Amersham Corp. NcoI and SphI were from
New England Biolabs, and BamHI and T4 DNA ligase were from
Promega. The oligonucleotide-directed in vitro mutagenesis
system, version 2.1, was a product of Amersham Corp. The magic
minipreps DNA purification system was a product of Promega.
Oligonucleotide synthesis and nucleotide sequence analysis were done by
the Iowa State University Nucleic Acid Facility. Ampicil-lin,
DL-dithiothreitol (DTT)(
)
,
phenylmethylsulfonyl fluoride, ATP, NADP, NADPH, glucose
1,6-bisphosphate, 1,5-anhydro-D-sorbitol, streptomycin,
phosphoenolpyruvate, deoxyribonuclease I (DNaseI), lactate
dehydrogenase/pyruvate kinase, and lysozyme were products of Sigma.
Isopropyl-1-thio-
-D-galactopyranoside (IPTG) was
pur-chased from Clontech. Bio-gel hydroxyapatite was obtained from
Bio-Rad Laboratories.
Expression of Mini-hexokinase pET-11d-mHKI
The
full-length cDNA for the human brain hexokinase I has been cloned into
the expression vector pET-11a in our laboratory
(32) . This
full-length cDNA clone was further digested with restriction enzyme
NcoI and BamHI to obtain a 1399-base pair
NcoI- BamHI fragment, which encodes a truncated form
of hexokinase starting from methionine-455 to the terminal amino acid.
The NcoI- BamHI fragment was then purified and ligated
into plasmid pET-11d, containing the bacteriophage T7 promoter,
previously digested with NcoI and BamHI to generate
the pET-11d-mHKI clone. pET-11d-mHKI was transformed into E. coli strain BL21(DE3), a phage -lysogen containing the T7 RNA
polymerase gene under control of the lacUV5 promoter. A 5-ml pregrowth
culture of the transformed strain in M9 medium plus 40 mg/liter
ampicillin and 4 mg/liter glucose was grown overnight and then added to
500 ml of the same medium. The culture was shaken at 37 °C to early
log phase ( A
= 0.4), and IPTG was then
added to a final concentration of 0.4 mM to induce the T7 RNA
polymerase gene. Cultures were shaken vigorously for an additional
24-40 h at 20 °C.
Purification of Mini-hexokinase and Full-length
Hexokinase
Protein purification was performed essentially as
described previously
(32) with some modifications. Cells were
harvested by centrifugation at 4000 g for 10 min and
washed once with 10 mM KP
, pH7.0. The resultant
pellet was resuspended in 120 ml of 5 mM KP
, pH
7.0, containing 1 mM EDTA, 50 mM glucose, 1
mM DTT, 9% glycerol, 2 mg/ml lysozyme, and 1 mM
phenylmethylsulfonyl fluoride. The cell suspension was kept at 0 °C
for 1 h and then sonicated four times for 20 s at 40 watts. To the
mixture, 600 µl of diluted DNase (150 µl of 10 mg/ml DNase
stock plus 650 µl of 1 M MgCl
) was added, and
the suspension was incubated at room temperature for 20 min. The lysate
was centrifuged at 17,500
g for 45 min. The
supernatant fluid was purified further by streptomycin,
(NH
)
SO
precipitation, heating, and
an ion exchange HPLC column step as described elsewhere
(32) .
The concentrated fractions with high enzyme activity were then loaded
onto a hydroxyapatite column(1
25 cm) previously equilibrated
with 25 mM potassium, pH7.0, containing 1 mM DTT. A
linear 360-ml gradient from 25 mM to 0.5 M KP
was used to elute mini-hexokinase. The fractions with high enzyme
activity were pooled, concentrated, and then injected onto Bio-Sil SEC
gel filtration HPLC columns (600
7.5 mm and 300
7.5 mm
connected together). Hexokinase activity was eluted with 50 mM
KP
buffer, pH 7.0, containing 0.1 M KCl and 1
mM DTT.
Site-directed Mutagenesis
Mutagenesis was
performed by following the instructions provided with the
oligonucleotide-directed in vitro mutagenesis system version
2.1 from Amersham Corp. The oligonucleotides used for mutagenesis are
as follows: 5`-ACC-AAT-TTC-ATT-GTG-CTG-CTG-3` for R539I,
5`-CTC-ATT-GTT-ATT-ACC-GGC-AGC-3` for G679I,
5`-TCA-TTT-CCC-TCC-CAG-CAG-ACG-3` for C606S, and
5`-GCA-ACA-GAC-TCC-GTG-GGC-CAC-3` for C628S, where the underlines
represent the mutated bases. The DNA sequence of the mutated gene was
verified by DNA sequencing using the chain termination method
(33) . After mutagenesis, the BamHI- SphI
fragment of the mini-hexokinase gene was cloned back into the
expression vector pET-11d, similarly digested with BamHI and
SphI, and the BamHI- BamHI fragment of
full-length hexokinase was cloned back into the expression vector
pET-11a, which is also digested with BamHI, creating
pET-11d-C606S, pET-11d-C628S, pET-11a-R539I, and pET-11a-G679I.
Hexokinase Activity Assay
Hexokinase activity was
determined spectrophotometrically as described previously
(32) .
Preparation of 1,5-An-G6P
1,5-An-G6P was prepared
as described previously
(34) .
Determination of Protein Concentration
Protein
concentration was determined by the method of Bradford
(35) ,
using bovine serum albumin as a standard.
Circular Dichroism Measurements
Circular Dichroism
(CD) spectra were recorded under the supervision of Dr. E. Stellwagon
at the University of Iowa. Measurements were made at room temperature
in a 0.1-cm path length cylindrical quartz cell. The concentration of
the sample was 0.15-0.66 mg/ml in 2 mM Hepes, pH 7.0,
containing 20 mM KCl and 0.2 mM mercaptoethanol. Base
lines were obtained using protein-free buffer solution.
Expression, Purification, and Characterization of
Wild-type Mini-hexokinase
E. coli strain BL21:DE3
(pET-mHKI) expresses a 50-kDa protein upon induction with IPTG.
Mini-hexokinase was purified from induced E. coli containing
pET-11d-mHKI to near homogeneity as described under ``Experimental
Procedures'' using SDS-polyacrylamide gel electrophoresis as the
criterion (data not shown). The specific activity of mini-hexokinase is
62, which is close to the value for full-length hexokinase from human
brain tissue
(32) . The Kvalues
of the substrates were determined to be 26 µM and 2.9
mM for glucose and ATP, respectively. Mini-hexokinase is
inhibited competitively relative to MgATP by glucose 1,6-bisphosphate
(Fig. 1). A cardinal feature of glucose-6-P-inhibited hexokinase
I is the ability of P
to reverse the inhibitory effects of
the product
(36, 37) . Experiments of glucose-6-P
inhibition and deinhibition by P
were carried out to
determine the effect of P
on the glucose-6-P-inhibited
mini-hexokinase. The results indicated that mini-hexokinase is
sensitive to inhibition by glucose-6-P, but 6 mM P
cannot relieve the inhibition caused by glucose-6-P. P
at 6 mM has little effect on mini-hexokinase in the
absence of glucose-6-P (Fig. 2 B). For full-length
hexokinase I, P
at 6 mM does ameliorate
glucose-6-P inhibition (Fig. 2 A)
(32, 38) . Therefore, we suggest that either the P
site resides in the N-terminal half of hexokinase I, or the
N-terminal half of hexokinase I is required for deinhibition
of glucose-6-P-inhibited hexokinase I.
Figure 1:
Inhibition of hexokinase I by
glucose-1,6-bisphosphate. A, full-length hexokinase I;
B, mini- hexokinase. The concentration of
glucose-1,6-bisphosphate was 0 (), 100 (+), and 167
µM (
). Activity was determined using the
glucose-6-phosphate dehydrogenase coupled assay. The measured change in
absorbance per min was plotted as percentage of the enzyme activity at
saturating concentrations of glucose and MgATP. The apparent K value for both mini-hexokinase and full-length hexokinase I is 100
µM.
Figure 2:
Effectiveness of inorganic phosphate in
reversing inhibition by 1,5-An-G6P. A, full-length hexokinase
I; B, mini-hexokinase. Concentration of glucose was 2
mM. Concentrations of 1,5-An-G6P and P were as
follows: 0 and 0 (
); 50 µM and 0 (+); 50
µM and 6 mM (
), respectively. OD,
optical density.
Site-directed Mutations of Arg , Gly in the Putative
ATP-binding Site
It has been reported that yeast hexokinase,
actin, and hsp70 heat shock proteins have similar three-dimensional
structures in their ATP-binding sites and, in addition, that the amino
acid residues are conserved in the ATP-binding sites
(29, 30, 31) . Based on this suggestion,
Arg and Gly
of human brain hexokinase might
be involved in ATP binding. The Arg
and Gly
residues were mutated to Ile by site-directed mutagenesis. Two
mutants, R539I and G679I, were created by site-directed mutagenesis.
The mutant R539I was purified to homogeneity and characterized
kinetically (). The k
value
decreased 114-fold, the K
value for ATP
increased 2-fold, and the K
value for
glucose decreased 4.6-fold. Little change was observed for the
K
values for 1,5-An-G6P. For the mutant
G679I, overexpressed hexokinase is insoluble. SDS-polyacrylamide gel
electrophoresis indicated that mutant G679I hexokinase was
overexpressed, but the cell extract did not contain hexokinase (data
not shown). The Gly
residue is probably important for
proper folding of the protein.
Site-directed Mutations of Cys and Cys in the
Putative Glucose-binding Site
Two mutants of mini-hexokinase
were prepared in order to investigate whether Cys and
Cys
are involved in glucose binding and/or catalysis.
Both mutants, C606S and C628S, exhibit 3-fold increases in
K
values for glucose compared with
wild-type mini-hexokinase; however, no alteration was observed on the
K
for ATP (). In addition,
both mutants are similar to wild-type mini-hexokinase in enzyme
activity. The CD spectra for mutants C606S and C628S and wild-type
mini-hexokinase show no differences (data not shown). These results
suggest that Cys
and Cys
are not involved
in catalysis and that covalent modification of Cys
and
Cys
by N-(bromoacetyl)-D-glucosamine
very probably caused inactivation by disrupting the native conformation
of brain hexokinase.
Effect of Mutations on the CD Spectra of Mini-hexokinase
and Full-length Hexokinase
To check whether the change in
Kvalues of mutants is due to gross
structural perturbations, CD experiments were carried out for the
wild-type enzyme, mini-hexokinase, and the mutants C606S, C628S, and
R539I. C606S and C628S mutants exhibit no differences in their CD
spectra compared with that of the wild-type mini-hexokinase (data not
shown). These results suggest that the mutant amino acid residues at
Cys
and Cys
do not cause changes in
secondary structure. R539I and wild-type hexokinase showed only a
slight difference in CD spectra (Fig. 3).
Figure 3:
CD spectra for wild-type hexokinase I
() and mutant D539I (
). The concentration of the sample
was adjusted to 660 µg/ml.
known to deinhibit glucose-6-P-inhibited
full-length hexokinase
(32, 38) (6 mM), no
deinhibition was observed. We conclude, therefore, that the allosteric
P
site proposed for brain hexokinase
(39) either
resides in the N-terminal half of hexokinase I or requires that portion
of the molecule for expression. However, White and Wilson
(27) claimed that P
ameliorates the inhibition of
mini-hexokinase by 1,5-An-G6P. The reason that our results are
different from Wilson's findings is not clear. The observation
that mini-hexokinase exhibits a 3-fold increase in
K
for ATP compared with the full-length
enzyme suggests that the truncated and native enzymes exhibit somewhat
different kinetic properties, e.g. the N-terminal region of
hexokinase I may be required for maintaining the proper conformation of
the active site.
on
hexokinase I is in some way associated with the N-terminal half of the
enzyme.
- and
-phosphates
of the nucleotide, shows sequence similarity. In this motif, positively
charged lysine or arginine residues form a salt bridge to the
negatively charged nucleotide phosphates. In brain hexokinase, the
corresponding residue is Arg
. In this report Arg
was mutated to Ile. The k
value for the
mutant decreased 114-fold relative to the wild-type enzyme. The
K
value for ATP increased 2-fold, whereas
the K
value for glucose decreased
4.6-fold. No change was observed for the K
value for 1,5-An-G6P. CD spectra showed only a minor change in
secondary structure. We suggest therefore that Arg
is
important for catalysis. Since the currently available yeast hexokinase
structure
(51) does not provide for an ATP site, we used actin
as a model for the ATP-binding site. In actin the residue corresponding
to Arg
is Lys
(29, 30, 31) . The
-amino group
interacts with the side chain carboxylate group of Asp
,
which binds to the metal ion. In the ADP-actin complex, the
-amino
group forms hydrogen bonds with the
- and
-phosphates. We
suggest by analogy to actin that Arg
is important for
catalysis by stabilizing the transition state product ADP-hexokinase
complex. In the motif PHOSPHATE 2, instead of the conserved Asp
of actin/hsc70 in which Asp
binds the metal ion,
brain hexokinase has Gly
. When Gly
was
mutated to Ile, the overexpressed mutant, G679I, was found to be
insoluble. It is possible that G679I is overexpressed in the form of
inclusion bodies. We suggest that Gly
is probably
important for the proper folding of the protein.
and the other
containing Cys
, was sensitive to the addition of
protective ligands, glucose, or N-acetylglucosamine. One
explanation for this observation is that sulfhydryl groups are
important for activity, contrary to the previous conclusion that only
one sulfhydryl group is critical. Another explanation is that neither
sulfhydryl group is important; however, modification of either
sulfhydryl by a large molecule, e.g. GlcNBrAc, will hinder the
binding of glucose. To determine the importance of Cys
and Cys
, Cys residues were mutated to Ser at these
two positions. Both of these mutants exhibited 3-fold increase in
K
values for glucose, but no change in
either the K
for ATP or the
k
was observed. These results suggested that
Cys
and Cys
are not critical residues for
either catalysis or binding of substrates. Amino acid sequence
comparisons between yeast hexokinases A and B and brain hexokinase type
I show no corresponding Cys residues in yeast hexokinase, which also
implies that these two Cys residues might not be important in the
functioning of hexokinase I. According to x-ray diffraction studies of
yeast hexokinase
(16, 17) , Cys
and
Cys
are located at or near the glucose-binding site.
Their modification with big molecules may hinder the entrance of the
substrate and lead to the loss of activity. Results of the current
investigation suggest that the x-ray diffraction crystallographic
structure of the ATP-binding sites of actin and hsp70
(29, 30, 31) are excellent models for the
ATP-binding site of human brain hexokinase based upon site-specific
mutagenesis and kinetic studies.
Table: A comparison of the kinetic data of the wild
type and the mutant forms of hexokinase
-D-galactopyranoside; 1,5-An-G6P,
1,5-anhydroglucitol 6-phosphate; DNase I, deoxyribonuclease I; HPLC,
high pressure liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.