From the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011
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ABSTRACT |
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Mutants of hexokinase I (Arg539
Lys, Thr661
Ala, Thr661
Val,
Gly534
Ala, Gly679
Ala, and
Gly862
Ala), located putatively in the vicinity of the
ATP binding pocket, were constructed, purified to homogeneity, and
studied by circular dichroism (CD) spectroscopy, fluorescence
spectroscopy, and initial velocity kinetics. The wild-type and mutant
enzymes have similar secondary structures on the basis of CD
spectroscopy. The mutation Gly679
Ala had little effect
on the kinetic properties of the enzyme. Compared with the wild-type
enzyme, however, the Gly534
Ala mutant exhibited a
4000-fold decrease in kcat and the
Gly862
Ala mutant showed an 11-fold increase in
Km for ATP. Glucose 6-phosphate inhibition of the
three glycine mutants is comparable to that of the wild-type enzyme.
Inorganic phosphate is, however, less effective in relieving glucose
6-phosphate inhibition of the Gly862
Ala mutant,
relative to the wild-type enzyme and entirely ineffective in relieving
inhibition of the Gly534
Ala mutant. Although the
fluorescence emission spectra showed some difference for the
Gly862
Ala mutant relative to that of the wild-type
enzyme, indicating an environmental alteration around tryptophan
residues, no change was observed for the Gly534
Ala and
Gly679
Ala mutants. Gly862
Ala and
Gly534
Ala are the first instances of single residue
mutations in hexokinase I that affect the binding affinity of ATP and
abolish phosphate-induced relief of glucose 6-phosphate inhibition,
respectively.
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INTRODUCTION |
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Hexokinase catalyzes the phosphorylation of glucose, using ATP as a phosphoryl donor. Four isoforms of hexokinase exist in mammalian tissue (1). Hexokinase isoforms I, II, and III have molecular weights of approximately 100,000 and are monomers under most conditions. Amino acid sequences of isoforms I-III are 70% identical (2). Moreover the N- and C-terminal halves of isoforms I-III have similar amino acid sequences, probably as a result of gene duplication and fusion (3-7). Hexokinase isoform IV (glucokinase) has a molecular weight of 50,000, similar to that of yeast hexokinase. Glucokinase exhibits (as does yeast hexokinase) significant sequence similarity to the N- and C-terminal halves of isoforms I-III.
Despite sequence similarities, the functional properties of hexokinase isoforms differ significantly. Isoform I (hereafter, brain hexokinase or hexokinase I) governs the rate-limiting step of glycolysis in brain and red blood cells (8, 9). The reaction product, glucose 6-phosphate (Glu-6-P1), inhibits both isoforms I and II (but not isoform IV) at micromolar levels. Inorganic phosphate (Pi), however, reverses Glu-6-P inhibition of only hexokinase I. The C-terminal domain of hexokinase I possesses catalytic activity, whereas the N-terminal domain is involved in the Pi-induced relief of product inhibition (10). In contrast, both the C- and N-terminal halves exhibit comparable catalytic activity in isoform II (11). Thus, among hexokinase isoforms, brain hexokinase exhibits unique regulatory properties in that physiological levels of Pi can reverse inhibition due to physiological levels of Glu-6-P (12-14).
The crystal structures of yeast hexokinase (15-17) are the basis for a model of mammalian glucokinase and its glucose binding site (18). The C-terminal domain of human brain hexokinase and its ATP binding site has been modeled in our laboratory based on similarities among the ATP-binding domains of actin, heat shock protein, and glycerol kinase (19). The model for the complex of ATP with hexokinase I puts a number of residues in the vicinity of ATP, of which Thr680, Asp532, and Arg539 have been the focus of directed mutations and investigations of initial rate kinetics (19, 20). These residues evidently stabilize the transition state, but do not influence the binding affinity of ATP (19, 20).
This study presents the results of CD, fluorescence, and kinetic
investigations of Gly534 Ala, Arg539
Lys, Thr661
Ala, Thr661
Val,
Gly679
Ala, and Gly862
Ala mutants of
brain hexokinase. The mutation Gly862
Ala causes an
order of magnitude increase in the Km for ATP, the
first instance of a mutation in hexokinase I that has had a significant
influence on the binding affinity of ATP. The mutation
Gly534
Ala reduces kcat by three
orders of magnitude, but more significantly abolishes
Pi-induced relief of Glu-6-P inhibition. Mutation at Gly534 is the first instance whereby a single mutation in
hexokinase I has had a significant impact on the amelioration of
product inhibition by Pi.
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EXPERIMENTAL PROCEDURES |
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Materials--
Affi-Gel Blue and Bio-gel hydroxyapatite came
from Bio-Rad. The TransformerTM site-directed mutagenesis
kit (2nd version) was a product of CLONTECH. The
Magic Minipreps DNA purification system was a product of Promega.
Oligonucleotide synthesis and nucleotide sequencing was done by the
Iowa State University nucleic acid facility. NruI and
XhoI were obtained from New England Biolabs and Promega,
respectively. The pET-11a plasmid was purchased from Novagen.
Escherichia coli strains BL21(DE3) and TG-1 were products of
Amersham Corp. E. coli strain ZSC13 were gifts of the
Genetic Stock Center, Yale University. Ampicillin, phenylmethylsulfonyl
fluoride, ATP, NADP, NADPH, glucose 1,6-bisphosphate, deoxyribonuclease
I, isopropyl-1-thio--D-galactopyranoside, and
1,5-anhydro-D-sorbitol were products of Sigma.
Expression of Human Brain Hexokinase--
The cDNA for human
hexokinase I was cloned into the expression vector pET-11a to generate
pET-11a-HKI (21). pET-11a-HKI was transformed into E. coli
strain ZSC13, which does not contain endogenous hexokinase. A 100-ml
culture of the transformed E. coli was grown overnight in LB
medium plus 40 mg/liter ampicillin and then added to 10 liters of the
same medium. The culture was grown in a fermentor at 37 °C to early
log phase (A600 = 0.4) after which
isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 0.4 mM to induce the T7 RNA
polymerase gene. The culture was grown with stirring (200 rpm) and a
filtered air flow of 5 p.s.i. for 24 h at 22 °C.
Purification of Wild-type and Mutant Brain
Hexokinase--
The wild-type and mutant forms of hexokinase were
purified as described elsewhere (20). The Gly862 Ala
mutant, however, did not bind to Affi-Gel Blue and was purified instead
by DEAE anion-exchange chromatography.
Site-directed Mutagenesis--
Mutagenesis was performed
by following the instructions provided with the
TransformerTM site-directed mutagenesis kit (2nd version)
from CLONTECH. The oligonucleotides used for
mutagenesis are as follows: 5-CTGGATCTTGCAGGAACC-3
for
Gly534
Ala, 5
-CTCATTGTTGCGACCGGC-3
for
Gly679
Ala, 5
-GTGGACGCGACACTCTAC-3
for
Gly862
Ala, 5
-CAGTGGGCGCAATGATGACC-3
for
Thr661
Ala, 5
-CAGTGGGCGTCATGATGACC-3
for
Thr661
Val, 5
-ACCAATTTCAAAGTGCTG-3
for
Arg539
Lys, where the altered codons are underlined.
The selective oligonucleotide sequence was chosen at the
NruI restriction site in the vector pET-11a and designed to
change the NruI site to a XhoI site. The sequence
of the selective oligonucleotide is 5
-CAGCCTCGCCTCGAGAACGCCAG-3
, where the underline
represents the XhoI site. Mutations were verified by
fluorescent dideoxy chain termination DNA sequencing.
Hexokinase Assay-- Hexokinase activity was determined spectrophotometrically as described previously (21). The kinetic parameters depicted in Table I were obtained from initial rate data obtained from two or more experiments. The substrate concentrations in the kinetic experiments were varied from Km/2 to 5Km. At least three concentrations of the inhibitor 1,5-anhydroglucitol-6-phosphate, from below to above its Ki, were used to evaluate its effect on the kinetics of the brain hexokinase enzyme.
Methods-- 1,5-anhydroglucitol-6-phosphate was prepared as described elsewhere (22). Protein concentration was determined by the method of Bradford (23) using bovine serum albumin as a standard. CD spectra were recorded using a Jasco J710 CD spectrometer as described elsewhere (20).
Fluorescence Emission Spectra--
The wild-type and mutant
enzymes were dialyzed against 20 mM Hepes buffer (pH 7.0)
containing 1 mM of -mercaptoethanol. The enzyme
concentration was 66.7 µg/ml. The fluorescent intensity was recorded
over the wavelength range from 300-350 nm, using an excitation
wavelength of 290 nm.
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RESULTS |
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Purification of the Wild-type and Mutant Human Brain
Hexokinase--
The use of a 10-liter fermentor enhanced enzyme yield
compared with 2-liter growth flasks. 20 liters of culture provided 70 mg of pure hexokinase I. The lack of retention of the
Gly862 Ala mutant on Affi-Gel Blue, a nucleotide
affinity column, is consistent with the elevated Km
for ATP exhibited by this mutant (see below). The wild-type and mutant
enzymes were more than 95% pure on the basis of SDS-polyacrylamide gel
electrophoresis (data not shown).
Characterization of Mutants by Kinetics and Spectroscopy--
The
kinetic parameters of wild-type and mutant enzymes are in Table
I. Compared with the wild-type enzyme,
the Gly534 Ala mutant showed a 4000-fold decrease in
kcat, and 4-, 5-, and 3-fold increases in the
Km (or Ki ) values for glucose,
ATP, and 1,5-anhydroglucitol-6-phosphate (an analog that mimics Glu-6-P
(24) and which can be used in the hexokinase-glucose-6-phosphate dehydrogenase coupled spectrophotometric assay), respectively. The
Gly862
Ala mutant showed an 11-fold increase in the
Km for ATP relative to the wild-type enzyme (Table
I), a 2-fold increase in the Km for glucose, an
18-fold decrease in kcat, but little change in
the Ki for 1,5-anhydroglucitol-6-phosphate. The
mutation of Gly679 to alanine did not change kinetic
properties, but the Gly679
Ile mutant was relatively
insoluble (20). Pi reverses Glu-6-P inhibition of the
Gly679
Ala mutant and the wild-type enzyme to the same
extent, whereas Pi has a modest or no effect on the
inhibition of the Gly862
Ala and Gly534
Ala mutants, respectively (Fig.
1).
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DISCUSSION |
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Arg539 putatively interacts with the polyphosphoryl
portion of ATP and stabilizes the transition state (19, 20). The
Arg539 Lys mutant is 10-fold more active than the
Arg539
Ile mutant (20), suggesting the importance of
the positive charge at position 539. However, as the Arg539
Lys mutant reported here is still 12-fold less active than wild-type hexokinase I, specific hydrogen bond interactions of the
arginyl side chain are of equal importance in stabilizing the
transition state. We have suggested, on the basis of previous work,
that Arg539 may form salt bridges with oxygen atoms of the
- and
-phosphoryl groups of ATP (19). The observed properties of
the Lys539 mutant are consistent with that suggestion.
Thr661 is 10 Å away from the -phosphoryl group of ATP
in our model, however, the
-oxygen atom of Thr661 is 3 Å from Asp657, which is putatively the catalytic base in
the abstraction of a proton from the 6-hydroxyl group of glucose (25).
The Thr661
Ala mutant has kinetic properties similar to
those of the wild-type enzyme, but mutation of Thr661 to
valine causes a 9-fold decrease in kcat,
probably by introducing an unfavorable nonbonded contact that perturbs
Asp657.
Consensus sequences, Gly-X-X-Gly-X-Gly-Lys-(Ser/Thr) in mononucleotide-binding proteins (26), Gly-X-Gly-X-X-Gly in dinucleotide-binding proteins (26), and Y-Gly-X-Gly-X-(Phe/Tyr)-Gly-X-Val, where Y is a hydrophobic residue for protein kinases (27), are rich in conserved glycines. In the mononucleotide-binding protein, p21H-ras, the dihedral angles of the polypeptide chain require glycine (28). For dinucleotide-binding proteins (29), the second glycine of the consensus sequence provides space for the polyphosphoryl moiety, and the first and third glycines satisfy conformational constraints of the polypeptide chain. The glycine-rich sequences of protein kinases participate in nucleotide binding, substrate recognition, and enzyme catalysis (30, 31). Yeast hexokinase, actin, hsc70, and glycerol kinase, however, are without a consensus sequence for nucleotide binding. Instead, the residues associated with nucleotide binding are scattered throughout the primary structure, but come together at single sites in the context of the folded polypeptide chains (32). Interestingly, the ATP binding domains of yeast hexokinase, actin, hsc70, and glycerol kinase are also rich in glycine residues.
We have probed the corresponding glycines by directed mutation, in the
expectation that some of these glycines are linked to observed kinetic
properties in hexokinase I. The mutation of Gly534 to
alanine produced a dramatic effect on kcat
(4000-fold reduction) and modest effects on Km for
glucose and ATP. Gly534 is conserved in sequences of
hexokinase, but according to our model (Fig.
3), its main chain torsion angles fall in
the allowed region of the Ramachandran plot for alanine. Instead, C
of Ala534 is 3.6 Å from an oxygen of the
-phosphoryl
group of ATP, but perhaps of greater significance is its 2.4 Å contact
with backbone carbonyl 537 of an adjacent
-strand. Our model
suggests then, the possibility of conformational change in the vicinity
of residue 534 to relieve the close contact mentioned above. Such a
local conformational change could influence Asp532, which
on the basis of earlier work (19) plays a critical role in the
stabilization of the transition state and may be involved in the
binding of Mg2+. A larger perturbation on the active site
due to the mutation of Gly534 to alanine is not likely,
because Km values for substrates are not influenced
and CD spectroscopy indicates no change in secondary structure.
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The Gly534 Ala mutant represents the first instance
whereby the mutation of a single residue has abolished
Pi-induced relief of Glu-6-P inhibition in hexokinase I. Glu-6-P inhibition of the C-terminal half of hexokinase I
(mini-hexokinase) cannot be reversed by Pi, implicating the
N-terminal domain in the relief of inhibition (20). The mechanism by
which Pi relieves Glu-6-P inhibition then evidently
involves structural elements of both the N- and C-terminal halves of
hexokinase I. Furthermore, the loss of Pi-induced relief of
Glu-6-P inhibition in the Gly534
Ala mutant is linked
closely to position 534, as mutations of Asp532 to lysine
and glutamate have little effect on this property (19).
Gly679 and Gly862 belong to reverse turns,
which pack against each other in our model (Fig. 3). The main chain
torsion angles put positions 679 and 862 in unallowed regions of the
Ramachandran plot for alanine ( =
111,
=
133 for
Gly679;
= 70,
= 168 for Gly862). Of the
two mutants, Gly862
Ala has the conformation of highest
energy. Although the C
atoms at positions 679 and 862 probably do
not interact with ATP, they make unfavorable contacts in our model with
backbone amide 863 (2.6 Å) and backbone amide 679 (2.7 Å),
respectively. These unfavorable contacts may not be significant,
however, as the mutation of Gly679 to alanine has no effect
on the kinetic properties of the enzyme. Instead, the introduction of
alanine at position 862 probably causes conformational changes in main
chain torsion angles. Although the fluorescence spectra of the
wild-type and Gly862
Ala enzymes differ (indicating a
perturbation in the local environment of tryptophan residues) their CD
spectra are identical (indicating no change in secondary structure).
Furthermore, the Ki for
1,5-anhydroglucitol-6-phosphate and the Km for
glucose are similar for the Gly682
Ala mutant and the
wild-type enzyme. Thus the mutation of Gly862 to alanine
probably has an effect only on residues in the vicinity of position
862. Thr863, a residue conserved in hexokinase sequences,
interacts with the ribose and base moieties of ATP in our model (Fig.
3). The Gly682
Ala mutant, then, could influence
interactions involving the base of ATP by perturbing the conformation
or relative position of Thr683. The mutation of
Gly862 to alanine increases the Km for
ATP by 11-fold without large changes in other kinetic parameters and as
such, represents the first mutation, which to our knowledge influences
the binding affinity of ATP.
Mutations prepared here and from previous studies (19) show a trend
that may be significant to the function of hexokinases in general.
Mutations of hexokinase I, which putatively influence interactions
involving the polyphosphoryl moiety of ATP, have no effect on
Km but a large effect on
kcat. Conversely, the Gly862 Ala
mutant, which putatively influences interactions at the base moiety,
has little effect on kcat but substantial
effects on Km for ATP. Interactions involving the
base of ATP are important for affinity, but polyphosphoryl-protein
interactions are involved in the stabilization of the transition state.
Conceivably, hexokinase I diverts energy from favorable interactions
between the polyphosphoryl moiety of ATP and the enzyme to promote
conformational changes that stabilize the transition state. This
phenomenon is not without precedence. In adenylosuccinate synthetase
from E. coli mutations involving protein interactions at the
base of GTP affect Km (33), whereas interactions
between the polyphosphoryl group of GTP and the protein (and
Mg2+) contribute to the stability of the transition state
by driving conformational changes in the active site (34).
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ACKNOWLEDGEMENT |
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We thank Laura Duck for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by research Grant NS 10546 from the National Institutes of Health and Grant MCB-9603595 from the National Science Foundation. This is Journal Paper J-17668 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project 3191.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Div. of Molecular Oncology, Washington University
School of Medicine, St. Louis, MO 63110.
§ Present address: Dept. of Microbiology, Shandong University, Jinan, Shandong 250100, People's Republic of China.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, 1210 Molecular Biology Bldg., Iowa State University, Ames, IA 50011. Tel.: 515-294-4971; Fax: 515-294-0453; E-mail: hjfromm{at}iastate.edu.
1 The abbreviation used is: Glu-6-P, glucose 6-phosphate.
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REFERENCES |
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