From the Department of Biochemistry and Biophysics, Iowa State
University, Ames, Iowa 50011
Crystal structures of human hexokinase I reveal
identical binding sites for phosphate and the 6-phosphoryl group of
glucose 6-phosphate in proximity to Gly87,
Ser88, Thr232, and Ser415, a
binding site for the pyranose moiety of glucose 6-phosphate in
proximity to Asp84, Asp413, and
Ser449, and a single salt link involving Arg801
between the N- and C-terminal halves. Purified wild-type and mutant
enzymes (Asp84
Ala, Gly87
Tyr, Ser88
Ala, Thr232
Ala,
Asp413
Ala, Ser415
Ala,
Ser449
Ala, and Arg801
Ala) were
studied by kinetics and circular dichroism spectroscopy. All eight
mutant hexokinases have kcat and
Km values for substrates similar to those of
wild-type hexokinase I. Inhibition of wild-type enzyme by
1,5-anhydroglucitol 6-phosphate is consistent with a high affinity
binding site (Ki = 50 µM) and a second, low affinity binding site (Kii
= 0.7 mM). The mutations of Asp84,
Gly87, and Thr232 listed above eliminate
inhibition because of the low affinity site, but none of the eight
mutations influence Ki of the high affinity site.
Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for
Asp84
Ala, Ser88
Ala,
Ser415
Ala, Ser449
Ala and
Arg801
Ala mutant enzymes is substantially less than
that of wild-type hexokinase and completely absent in the
Gly87
Tyr and Thr232
Ala mutants. The
results support several conclusions. (i) The phosphate regulatory site
is at the N-terminal domain as identified in crystal structures. (ii)
The glucose 6-phosphate binding site at the N-terminal domain is a low
affinity site and not the high affinity site associated with potent
product inhibition. (iii) Arg801 participates in the
regulatory mechanism of hexokinase I.
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INTRODUCTION |
Hexokinase (ATP:D-hexose 6-phosphotransferase, EC
2.7.1.1) catalyzes the phosphorylation of the 6-hydroxyl of glucose,
using ATP as a phosphoryl donor. Brain hexokinase (hereafter hexokinase I) is one of four isoforms found in mammalian tissue. In brain tissue
and red blood cells hexokinase I participates in the regulation of
glycolysis (1-5). Hexokinase IV (glucokinase) has properties similar
to that of yeast hexokinase. It consists of a single polypeptide chain
of molecular mass 50 kDa and is not inhibited by physiological levels
of glucose 6-phosphate
(Glu-6-P).1 Hexokinase
isoforms I, II, and III have polypeptide chains of molecular mass 100 kDa. Glu-6-P is a potent inhibitor of isoforms I-III (5), but only for
hexokinase I does phosphate (Pi) relieve Glu-6-P inhibition
(6-8). Isoforms I-III have 70% identical amino acid sequences.
Furthermore, the N- and C-terminal halves of isoforms I-III have
similar amino acid sequences, putatively a consequence of the
duplication and fusion of a primordial hexokinase gene (9-12).
The isolated C-terminal half of hexokinase I is catalytically active,
whereas the N-terminal half by itself or in the context of the
full-length protein has no catalytic activity (13-15). On the other
hand, the N-terminal half of hexokinase I is involved in the
Pi-induced relief of product inhibition on the basis of the
following. (i) The isolated C-terminal half loses the property of
Pi-induced relief of Glu-6-P inhibition (13, 16, 17). (ii)
A chimeric hexokinase containing the N-terminal half of hexokinase I
and C-terminal half of hexokinase II exhibits Pi-induced
relief of Glu-6-P inhibition (18). (iii) The crystallographic structure of recombinant hexokinase I complexed with glucose and phosphate reveals a single site for phosphate, that being at the N-terminal half
of the enzyme (19). Two different mechanisms of regulation, however,
have been proposed by investigators. In one, Glu-6-P binds to the
N-terminal half as an allosteric inhibitor and Pi competes
directly with Glu-6-P. In the other, Pi binds to the N-terminal half and displaces Glu-6-P from the active site (C-terminal half) by way of an indirect mechanism. In both models the binding of
phosphate to the N-terminal domain regulates catalysis at the C-terminal half by way of an allosteric mechanism.
Hexokinase I is a dimer in its crystalline complex with glucose and
Glu-6-P (20). Glu-6-P and glucose bind at both the C- and N-terminal
halves, and both halves adopt closed conformational states. The
structure of a glucose-Pi complex of hexokinase I is also a
dimer (19). Gluose and Pi bind at the N-terminal half only,
and the C- and N-terminal halves are in opened and closed conformational states, respectively. Pi and the
6-phosphoryl group of Glu-6-P bind to the same residues at the
N-terminal half, whereas the pyranose moiety of Glu-6-P binds at a
separate locus. Gly87, Ser88,
Thr232, and Ser415 are at the
phosphate/6-phosphoryl binding site. Asp84,
Asp413, and Ser449 interact with the pyranose
moiety of Glu-6-P. Contact between the N- and C-terminal halves of
hexokinase I occurs only by way of a transition helix and a salt
link involving Arg801 (19, 20).
Presented here are the functional properties of wild-type and mutant
hexokinases (Asp84
Ala, Gly87
Tyr,
Ser88
Ala, Thr232
Ala,
Asp413
Ala, Ser415
Ala,
Ser449
Ala and Arg801
Ala).
Gly87
Tyr and Thr232
Ala separately
abolish Pi relief of inhibition caused by the product
analog 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). Hence the site
for Pi binding identified by crystallographic
investigations is most likely the Pi regulatory site of
hexokinase I. Kinetic data from the wild-type enzyme infer the presence
of high and low affinity inhibitory sites for 1,5-AnG-6-P.
Asp84
Ala, Gly87
Tyr, and
Thr232
Ala eliminate inhibition due to the low affinity
site with no effect on the high affinity site. Glu-6-P interactions at
the N-terminal half then are correlated with the low affinity binding site observed in kinetics. Finally, mutation of Arg801
reduces Pi relief of product inhibition and inhibition
associated with the low affinity site. Arg801 evidently
plays a role in the transmission of the allosteric signal from the
N-terminal half to the active site of hexokinase I.
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EXPERIMENTAL PROCEDURES |
Materials--
A full-length cDNA of human brain hexokinase,
cloned into an expression vector pET-11a (from Novagen) to produce
pET-11a-HKI, was available from a previous study (21). The Altered
Sites II in vitro Mutagenesis System, T4 polynucleotide
kinase, T4 DNA ligase, plasmid pGEM-7Z(+), and restriction enzymes were
from Promega. Bio-Gel hydroxyapatite came from Bio-Rad. Oligonucleotide synthesis and DNA sequencing were done at the Iowa State University Nucleic Acid Facility. Escherichia coli strain ZSC13, which
does not contain endogenous hexokinase, was a gift from the Genetic Stock Center, Yale University. ATP, NADP, streptomycin sulfate, glucose
1,6-bisphosphate (Glu-1,6-P2),
1,5-anhydro-D-sorbitol, deoxyribonuclease I (DNase I),
leupeptin, phenylmethylsulfonyl fluoride, and ampicillin were from
Sigma. Glucose-6-phosphate dehydrogenase came from Boehringer Mannheim.
Isopropyl-1-thio-
-D-galactopyranoside (IPTG) was from
BioWorld.
Construction of Mutant Hexokinase Genes--
The hexokinase gene
was mutated according to the protocols of the Promega Altered Sites II
in vitro Mutagenesis System with only slight modification.
The pALTER-Ex1 vector from the mutagenesis system was
modified first by inserting a small XbaI-BamHI
fragment from the plasmid pGEM-7Z(+) to introduce a ClaI
site. An XbaI-ClaI fragment, containing the
hexokinase cDNA of pET-11a-HKI, was inserted then into the modified
pALTER-Ex1 vector. The resulting construct served as the DNA
template in mutagenesis work. The oligonucleotide primers used in
site-directed mutations were 5'-TTC ATT GCC CTG GCT CTT GGT
GG-3' (Asp84
Ala), 5'-C CTG GAT CTT GGT TAC
TCT TCC TTT CGA-3' (Gly87
Tyr), 5'-GAT CTT GGT GGG
GCT TCC TTT CGA-3' (Ser88
Ala), 5'-CTG ATC
ATC GGC GCT GGC ACC AAT-3' (Thr232
Ala),
5'-CG GTT GGT GTC GCA GGA TCT CTT TAC AA-3'
(Asp413
Ala), 5'-GGT GTC GAC GGA GCA CTT TAC
AAG ACG-3' (Ser415
Ala), 5'-GAG AGT GGC GCA
GGC AAG GGG GCT-3' (Ser449
Ala), and 5'-CTG CTC CAG GTC
GCT GCT ATC CTC CAG-3' (Arg801
Ala), where
the altered codons are in bold type. For mutations, Asp84
Ala, Gly87
Tyr, Ser88
Ala,
Thr232
Ala, Asp413
Ala,
Ser415
Ala, and Ser449
Ala, the
XbaI-NcoI fragment carrying the gene for the
mutated N-terminal half of hexokinase was ligated to the 7-kb
XbaI-NcoI fragment of pET-11a-HKI to reconstruct
the expression vector pET-11a-HKI. For the mutation Arg801
Ala, the XbaI-ClaI fragment carrying the
whole gene of mutated hexokinase was ligated to the 5.5-kb
XbaI-ClaI fragment of pET-11a-HKI to reconstruct
the expression vector pET-11a-HKI. The whole
XbaI-NcoI fragment carrying the mutated gene for
the N-terminal domain or the XbaI-ClaI fragment
carrying the mutated whole gene of hexokinase was sequenced to confirm
the presence of only the desired mutation.
Expression and Purification of Wild-type and Mutant Hexokinase
I--
Wild-type and mutant forms of hexokinase I were produced by
growing pET-11a-HKI-transformed E. coli strain ZSC13. The
pET-11a-HKI-transformed E. coli was grown overnight in LB
medium plus 40 µg/ml ampicillin and then added to a 100-fold volume
of M9 medium with 100 µg/ml ampicillin. The culture was grown in a
fermentor (with stirring of 200 rpm and a filtered air flow of 5 p.s.i.) or in flasks (with shaking at 220 rpm) at 37 °C to early log
phase (A600 = 0.4), after which IPTG was added
to a final concentration of 0.4 mM to induce the T7 RNA
polymerase gene. The culture was grown for an additional 20-24 h at
22 °C. The wild-type and mutant forms of hexokinase were purified by
using streptomycin sulfate precipitation, DEAE-anion exchange column
chromatography, hydroxyapatite column chromatography, and DEAE-5PW
Spherogel TSK-G HPLC chromatography as described elsewhere (17,
19-22).
Measurement of Protein Concentration--
The concentrations of
wild-type and mutant hexokinases were determined by the method of
Bradford (23), using bovine serum albumin as a standard.
Preparation of 1,5-Anhydroglucitol-6-phosphate--
The barium
salt of 1,5-AnG-6-P was prepared as described elsewhere (24). A
precisely weighed amount of the barium salt of 1,5-AnG-6-P was
dissolved in distilled water by adding HCl. Barium ion was removed by
precipitation with excess 1 M
Na2SO4 followed by centrifugation.
Hexokinase Activity Assay and Kinetic Studies--
Hexokinase
activity was determined by the glucose-6-phosphate
dehydrogenase-coupled spectrometric assay as described elsewhere (21).
Initial rate data were analyzed by using a computer program written in
MINITAB language with an
value of 2.0 (22). The concentration of
hexokinase used for activity assays was 1.2 ~ 2.0 µg/ml.
Circular Dichroism Measurements--
CD spectra were measured
from 200 to 260 nm at room temperature by using a J710 CD spectrometer.
The concentration of hexokinase used was 120 ~ 200 µg/ml in
12.5 mM Hepes buffer, pH 7.6, containing 0.125 mM glucose and 0.25 mM
-mercaptoethanol.
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RESULTS |
Purity of Wild-type and Mutant Hexokinase I--
The purity of
wild-type and mutant hexokinases is greater than 95% as judged by
SDS-polyacrylamide gel electrophoresis (Fig. 1).

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Fig. 1.
SDS-polyacrylamide gel electrophoresis of
purified wild-type and mutant hexokinases. A 7.5% SDS minigel was
stained with Coomassie Brilliant Blue R-250. Lanes
1-10 represent, respectively protein molecular weight
standards, wild-type hexokinase, Asp84 Ala,
Gly87 Tyr, Ser88 Ala,
Thr232 Ala, Asp413 Ala,
Ser415 Ala, Ser449 Ala, and
Arg801 Ala mutant hexokinases.
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Secondary Structure Analysis--
CD spectra of wild-type and
mutant hexokinases were essentially identical (data not shown),
indicating the absence of long range conformational changes as a
consequence of mutation.
Kinetic Studies--
Relative to the wild-type enzyme, all eight
mutant hexokinases exhibited similar kcat and
Km values (Table I).
As hexokinase activity is determined by the oxidation of Glu-6-P, inhibition studies must employ closely related analogs (1,5-AnG-6-P and
Glu-1,6-P2), which are chemically inert under the
conditions of assay. The analogs putatively bind to the same inhibitory
site as does Glu-6-P, albeit with slightly altered affinities.
1,5-AnG-6-P and Glu-1,6-P2 (up to concentrations of 100 and
334 µM, respectively) are linear competitive inhibitors
(with respect to ATP) of the wild-type enzyme and the mutant
hexokinases (data not shown). Mutations have no major effect on
Ki values for 1,5-AnG-6-P and Glu-1,6-P2
(Table I).
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Table I
Kinetic parameters for wild-type and mutant hexokinases
Reported values are the mean ± S.D. 1,5-AnG-6-P and
Gluc-1,6-P2 are the competitive inhibitors of hexokinase I
(versus ATP). The Ki was obtained from
plots of 1/velocity versus 1/[ATP] at 2 mM
glucose with inhibitor concentrations ranging from 0 to 100 µM and 0 to 334 µM for 1,5-AnG-6-P and
Gluc-1,6-P2, respectively.
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Crystallographic investigations employed millimolar concentrations of
Glu-6-P, and under such conditions the N- and C-terminal halves of
hexokinase I each bind one molecule of Glu-6-P. At concentrations of
1,5-AnG-6-P up to 100 µM, kinetic data support only one
functional binding site for the product analog (Table I). At
concentrations significantly above 100 µM, however,
inhibition of the wild-type enzyme by 1,5-AnG-6-P is nonlinear (Fig.
2). A model for linear competitive
inhibition (single high affinity site) accounts poorly for the data of
Fig. 2 (goodness-of-fit, 10.9%). A modified rapid equilibrium Random
Bi Bi mechanism (7), which incorporates two inhibitory sites for
Glu-6-P (Scheme I), is the simplest model that can account for the data of Fig. 2. In Scheme I, A, B,
Q, and P are ATP, glucose, Glu-6-P, and ADP,
respectively, bound productively to the C-terminal domain, I
is 1,5-AnG-6-P bound to inhibitory sites, E is hexokinase I,
and Kia, Kib, Ka, Kb, Ki,
and Kii are dissociation constants. Scheme I
presents equilibria between complexes which are pertinent to the
conditions of kinetic investigations presented here. 1,5-AnG-6-P can
interact with the free enzyme and with enzyme-ATP and enzyme-product
complexes, but under conditions of saturating glucose and initial
velocity protocols, these more complex schemes yield the same velocity
relationship as does Scheme I. Equation 1, developed from Scheme I
under conditions of saturating glucose and initial velocity protocols,
is below:
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(Eq. 1)
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In Equation 1, v represents the velocity,
Vm the maximum velocity, A the
concentration of ATP, I the concentration of 1,5-AnG-6-P,
Ka the dissociation constant for A from the EAB complex, Kb the dissociation
constant for B from the EAB complex,
Kia the dissociation constant for the EA
complex, Ki the dissociation constant for
I from the EBI complex, and
Kii the dissociation constant for I from
the EBII complex.

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Fig. 2.
Inhibition of wild-type hexokinase I
(A) and the Asp84 Ala
mutant (B) by 1,5-AnG-6-P. Concentrations of
1,5-AnG-6-P are 0 ( ), 100 ( ), 200 ( ), 300 ( ), 500 ( ),
700 ( ), and 1000 ( ) µM. Initial velocity
(V) was determined by the coupled spectrometric assay. OD is
optical density.
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Equation 1 accounts for the wild-type data with a goodness-of-fit 2.2%
and was used to fit inhibition data for all mutants (Table
II). However, Kii
adopted negative values for Asp84
Ala,
Gly87
Tyr, and Thr232
Ala, and the
reduction in the goodness-of-fit value for a two-site, relative to a
single-site model did not validate the determination of
Kii. Hence for Asp84
Ala,
Gly87
Tyr, and Thr232
Ala we report the
complete loss of inhibitory function associated with the low affinity
site. Indeed, the relevant plots for the Asp84
Ala
mutant, for instance, are consistent with a model of linear competitive
inhibition up to a concentration of 1 mM 1,5-AnG-6-P (Fig.
2).
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Table II
1,5-AnG-6-P inhibition constants for wild-type and mutant
hexokinase I
The values shown are the mean ± S.D. 1,5-AnG-6-P is the
competitive inhibitor of hexokinase I (versus ATP).
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The Pi-induced relief of Glu-6-P inhibition was studied at
two different concentrations of ATP and at several concentrations of
1,5-AnG-6-P and Pi. Mutations, Gly87
Tyr
and Thr232
Ala, abolish the relief of Glu-6-P
inhibition by Pi (Figs. 3 and
4). Mutations, Asp84
Ala, Ser88
Ala,
Ser415
Ala, and Arg801
Ala, reduce the
effect of Pi on Glu-6-P inhibition by approximately one-half relative to the wild-type enzyme (Fig.
4).

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Fig. 3.
Plot of (relative velocity of hexokinase
I) 1 versus 1,5-AnG-6-P concentration for
wild-type (A), Gly87 Tyr
(B), and Thr232 Ala
(C) hexokinases. The ATP concentration is 1.25 mM, and Pi concentrations are 0 ( ), 3 ( ),
and 6 ( ) mM. The relative velocity of hexokinase I in
the absence of 1,5-AnG-6-P and Pi is unity.
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Fig. 4.
Relief of 1,5-AnG-6-P inhibition induced by
Pi in wild-type and mutant hexokinases. Concentrations
of ATP and Pi, respectively, are 0.625 and 3 mM
(gray), 0.625 and 6 mM (hatched),
1.25 and 3 mM (black), and 1.25 and 6 mM (white). The concentrations of 1,5-AnG-6-P
are those given in Fig. 3. Relief of 1,5-AnG-6-P inhibition (%)
induced by Pi is defined as 100 × (A B)/A where A is the slope from plot
of (relative velocity of hexokinase I) 1 versus
1,5-AnG-6-P concentration (as in Fig. 3) in the absence of
Pi and B is the slope from plot of (relative
velocity of hexokinase I) 1 versus 1,5-AnG-6-P
concentration in the presence of Pi. For mutant
Asp84 Ala, Asp413 Ala, and
Ser449 Ala only one concentration of ATP (1.25 mM) has been used.
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DISCUSSION |
Crystal structures of recombinant human hexokinase I reveal a
bound phosphate or phosphoryl group at the N-terminal half of the
enzyme in the vicinity of Gly87, Ser88,
Thr232, and Ser415 (19, 20). The hydroxyl
groups of Ser88, Thr232, and Ser415
hydrogen-bond to phosphate in the glucose-Pi crystalline
complex and to the 6-phosphoryl group of Glu-6-P in the glucose-Glu-6-P complex (Fig. 5). In addition, a bulky
side chain at Gly87 should block the binding of
Pi or the phosphoryl group of Glu-6-P. Residue 87 can
accommodate bulky side chains without a significant perturbation to its
backbone conformation. The mutations Gly87
Tyr,
Ser88
Ala, Thr232
Ala and
Ser415
Ala then should either weaken or completely
block the interaction between the N-terminal half and phosphoryl
groups. On the other hand, mutations of Asp84,
Asp413, and Ser449 to alanine should disrupt
interactions between the pyranose moiety of product analogs and the
N-terminal half of hexokinase I.

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Fig. 5.
Stereoview of ligand binding sites at
N-terminal half of recombinant human hexokinase I. A,
interactions involving Pi in the glucose-Pi
complex (19). B, interactions involving Glu-6-P in the
glucose-Glu-6-P complex (20). Dashed lines
represent donor-acceptor interactions. Drawing was done with MOLSCRIPT
(28).
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The findings here can be reconciled easily with one of two models for
regulation of hexokinase I. The models differ primarily in the location
of the functional site for Glu-6-P inhibition. Either Glu-6-P binds to
the active site of the C-terminal half of the enzyme and competes
directly with ATP or Glu-6-P binds to the N-terminal half and inhibits
catalysis through an allosteric mechanism. Product analogs interact
equally well with the high affinity site of mutant hexokinases and the
wild-type enzyme (Table II), a result consistent with the direct
inhibition of the active site by Glu-6-P and inconsistent with the
allosteric model for Glu-6-P inhibition. The presence of bound Glu-6-P
at the N-terminal half in the crystal structure of the complex of
hexokinase I with glucose and Glu-6-P probably stems from the high
concentration of inhibitor employed in the growth of that crystal form
and the effects of dimerization at elevated protein concentrations.
Indeed, kinetic data here show clearly the presence of a low affinity, inhibitory site which is sensitive to mutations at the Glu-6-P pocket
of the N-terminal half of hexokinase I. As Glu-6-P stabilizes dimers of
hexokinase I, the additional inhibition associated with the low
affinity site may be a consequence of dimerization of the enzyme in the
presence of high concentrations of product analog (25).
Previously reported mutations of the N-terminal half (Asp84
Asn (26), Ser155
Ala (15), and Asp209
Ala (15)) involve residues that bind to the pyranose group of
Glu-6-P, as observed in the glucose/Glu-6-P crystal structure. Consistent with the properties of mutants reported here, mutations of
the pyranose region of the Glu-6-P binding pocket of the N-terminal half of the enzyme have little effect on catalysis or the inhibition of
catalysis. Previous experiments in kinetics, however, did not employ
high concentrations of inhibitor so no data are available regarding the
effects of these mutations on the low affinity inhibitory site.
Mutations of the Glu-6-P pocket of the C-terminal half of the enzyme
(Asp532
Asn (26), Asp532
Glu (22),
Asp532
Lys (22), Gly534
Ala (27),
Ser603
Ala (14), Asp657
Ala (14),
Thr680
Ser (22), Thr680
Val (22),
Gly862
Ala (27) and Gly896
Val (26)),
also have little effect on inhibition by product analogs. The mutations
in the C-terminal half of hexokinase I listed above generally cause a
significant loss of activity, and to compensate for low activity,
enzyme concentrations in assays were increased in some instances to
levels at which hexokinase I could dimerize. Hence, reported kinetic
parameters (22, 27) may reflect the properties of mutant dimers rather
than mutant monomers. Further information is necessary regarding the
state of aggregation of the C-terminal half mutants of hexokinase I before structure/function correlations can be made with certainty.
The results here directly confirm a functional role for the phosphate
binding site observed in the glucose/Pi crystalline complex. Seven hydrogen bonds hold Pi in place at the
N-terminal half of hexokinase I. The individual mutations of
Ser88, Thr232, and Ser415 to
alanine remove to a first approximation only one hydrogen bond, leaving
the remaining six in place. Depending on the contribution of that
hydrogen bond to the stability of the Pi-enzyme complex, one finds significant (up to 100%) but variable reductions in the
relief of product inhibition. On the basis of modeling studies, the
mutation of Gly87 to tyrosine would place the tyrosyl side
chain at the Pi site, potentially disrupting all seven of
its hydrogen bonds with phosphate, and indeed, Gly87
Tyr abolishes relief of product inhibition altogether.
The mutation Gly534
Ala reportedly reduces
Pi relief of product inhibition (27). Upon resequencing the
gene for the Gly534 mutant, however, a second site mutation
was detected at position 86. Hence the kinetic parameters reported for
the Gly534
Ala mutant are those of the double mutant
Gly86
Ala/Gly534
Ala. The observed
effect on the Pi relief of product inhibition is probably a
consequence of the mutation of Gly86 to alanine.
One of the goals of the present study was to ascertain whether the
crystalline hexokinase I dimers provide information regarding the
structure-function properties of the isolated monomer in solution. Crystal structures indicate a single Pi binding site at the
N-terminal domain, and on the basis of studies here, that site is
indeed the functional regulatory site for Pi. Crystal
structures also indicate the binding of Glu-6-P to the same site, and
indeed the results here are consistent with a low affinity site for
Glu-6-P at the N-terminal domain, which overlaps the functional
regulatory site for Pi. The participation of
Arg801 in a salt link with Asp251 and
Glu252 stands as the only noncovalent interaction between
the N- and C-terminal halves of hexokinase I (Fig.
6). Arg801 is the best
candidate on the basis of crystal structures for the transmission of
conformational changes in the N-terminal half to the active site. As a
50% reduction in the Pi relief of product inhibition
attends the mutation of Arg801 to alanine,
Arg801 is a likely participant in the allosteric mechanism
of hexokinase I regulation. Other residues, however, also must
participate in parallel with Arg801 in the allosteric
regulatory mechanism of hexokinase I. Otherwise the mutation of
Arg801 to alanine should abolish the Pi relief
of product inhibition entirely. Interestingly, the residue
corresponding to position 801 in hexokinase II is also an arginine,
consistent with the appearance of phosphate-linked phenomena in the
chimeric enzyme consisting of C-terminal half hexokinase II and
N-terminal half hexokinase I (18). The results presented here regarding
Arg801, the regulatory site for Pi, and a low
affinity site for Glu-6-P suggest that the crystal structures (19, 20)
provide reasonable approximations of the free monomer in solution.

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Fig. 6.
Overview of one of two polypeptide chains of
the glucose-Pi complex (19) showing the location of the
Arg801 salt link. The single glucose molecule bound to
the N-terminal domain near Pi is omitted for clarity.
Black circles represent sites for bound metal
cations. Drawing was done with MOLSCRIPT (28).
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