From the Departments of Molecular Physiology and
Biophysics and § Medicine, Vanderbilt University School of
Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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Mammalian hexokinases (HKs) I-III are composed
of two highly homologous ~50-kDa halves. Studies of HKI indicate that
the C-terminal half of the molecule is active and is sensitive to
inhibition by glucose 6-phosphate (G6P), whereas the N-terminal half
binds G6P but is devoid of catalytic activity. In contrast, both the N-
and C-terminal halves of HKII (N-HKII and C-HKII, respectively) are
catalytically active, and when expressed as discrete proteins both are
inhibited by G6P. However, C-HKII has a significantly higher
Ki for G6P (KiG6P)
than N-HKII. We here address the question of whether the high
KiG6P of the C-terminal half (C-half)
of HKII is decreased by interaction with the N-terminal half (N-half)
in the context of the intact enzyme. A chimeric protein consisting of
the N-half of HKI and the C-half of HKII was prepared. Because the
N-half of HKI is unable to phosphorylate glucose, the catalytic
activity of this chimeric enzyme depends entirely on the C-HKII
component. The KiG6P of this chimeric enzyme is similar to that of HKI and is significantly lower than that
of C-HKII. When a conserved amino acid (Asp209) required
for glucose binding is mutated in the N-half of this chimeric protein,
a significantly higher KiG6P (similar to that of C-HKII) is observed. However, mutation of a second conserved
amino acid (Ser155), also involved in catalysis but not
required for glucose binding, does not increase the
KiG6P of the chimeric enzyme. This resembles the behavior of HKII, in which a D209A mutation results in an
increase in the KiG6P of the enzyme,
whereas a S155A mutation does not. These results suggest an interaction in which glucose binding by the N-half causes the activity of the
C-half to be regulated by significantly lower concentrations of
G6P.
Hexokinases (ATP:D-hexose 6-phosphotransferase, EC
2.7.1.1; HK)1 catalyze the
phosphorylation of glucose to glucose 6-phosphate (G6P). Type I, II,
and III isozymes of mammalian HKs are ~100 kDa in size and are
inhibited by the reaction product, G6P. Type IV HK (also known as
glucokinase) is similar to yeast HK in that it has a relative mass of
~50 kDa and is insensitive to inhibition by physiologic
concentrations of G6P. The deduced amino acid sequences of HKI-III
reveal internal similarities between their N- and C-terminal halves and
between each of these and yeast HK and glucokinase (1). This
observation supports the hypothesis that the 100-kDa mammalian HKs
evolved from the duplication and fusion of an ancestral 50-kDa HK (1,
2).
The N- and C-terminal halves of HKI show a marked functional difference
despite the similarity of their amino acid sequences. The C-terminal
half of HKI is catalytically active, whereas the N-terminal half is
inactive (3-5). Whereas G6P binds to both halves of HKI (3, 5), the
G6P regulatory site of HKI is thought to be in the N-half of the intact
enzyme, and the C-half binding site is latent (1, 6). In contrast, we
have shown that both the N- and C-terminal halves of human and rat HKII
(N-HKII and C-HKII, respectively) have catalytic activity and that each is inhibited by G6P (7). However, the N- and C-terminal halves of HKII
possess different kinetic characteristics: N-HKII has a slightly higher
affinity for ATP than does C-HKII, and its Ki for
G6P is about 20-30-fold lower than that of C-HKII (7). Furthermore,
the KiG6P value of the intact 100-kDa
HKII enzyme is similar to that of N-HKII (7), an observation confirmed by Tsai and Wilson (8).
Based on the crystal structure of yeast HK (9-12) and recently
confirmed by a similar analysis of mammalian HKI (13-16), glucose is
thought to bind to a cleft in the open conformation of the enzyme. This
binding results in closure of the cleft and initiates the catalytic
sequence. Asp211 is an important residue in yeast
hexokinase. As the base catalyst, it binds the 6-hydroxyl group of
glucose, and this receives the phosphate moiety of ATP. Mutation of the
corresponding residue to alanine in glucokinase (D205A) (17), the
C-half of HKI (D209A) (18), and the N- and C-halves of HKII (D209A and
D657A) (7) results in a complete loss of activity. In contrast, the
carbonyl group of Ser158 in yeast HK interacts with the
3-hydroxyl group of glucose only in the closed conformation (1).
Mutation of the corresponding amino acid to alanine in the C-half of
HKI (S155A) (5) and the N- and C-halves of HKII in the intact enzyme
(S155A and S603A) reduces the Vmax of the enzyme
to about 10% of that of the wild type
(8).2 Thus, although the
D209A mutation in mammalian HKs results in a loss of glucose binding
and catalytic activity, the S158A mutation maintains glucose binding
but reduces the Vmax.
Because the KiG6P of HKII is similar to
that of N-HKII and significantly different from that of C-HKII, we
focused the present analysis on how the activity of C-HKII is regulated by G6P in the intact enzyme. Mutations that have different effects on
glucose binding in the N-half of HKII result in different valves of
KiG6P. We here show that the regulation
of the C-half by G6P in the intact HKII is influenced by the N-half of
the molecule.
Construction of NICII Chimeric Proteins--
The human
hexokinase I cDNA was provided by Dr. Graeme Bell (Howard Hughes
Medical Institute, University of Chicago). A DNA fragment that encodes
the N-half of HKI was prepared by PCR using B2392
(5'-GCCAGCATGATCGCCGCGCA-3') as the 5' oligonucleotide and A2670
(5'-TTACTGCCGGTGCTGCTGGG-3') as the 3' oligonucleotide. A2670 contains
a stop codon at position 1407, which is the putative fusion site
between the N- and C-terminal halves of HKI. The pGef plasmid (7),
constructed by insertion of a bacteriophage f1 origin into the pGex-3x
vector (Amersham Pharmacia Biotech), was linearized at a
SmaI restriction site located adjacent to the glutathione
S-transferase (GST) open reading frame. The PCR product that
contains the N-half of HKI was then ligated into pGef to obtain
pGST·N-HKI. This plasmid encodes amino acids 1-469 of human HKI
fused to the C terminus of GST.
An NcoI site is conveniently located near the fusion site of
the N- and C-terminal halves of both human HKI and HKII.
NcoI cleaves at nucleotide 1366 in the open reading frame of
both HKI and HKII. pGST·N-HKI was digested with NcoI and
BssHII (a cleavage site about 1.2 kbp 5' of the GST start
codon) to produce a fragment of ~3.2 kbp that contains the coding
sequence of GST and the N-terminal half of HKI. This fragment was
purified by size separation through agarose gel electrophoresis and was
isolated using Spin-x tubes (Costar, Cambridge, MA). A GST·HKII
expression plasmid, pGST·HKII (7), was also digested with
NcoI and BssHII, and the larger fragment of about
5.2 kbp was isolated. These two fragments were ligated together to
generate the plasmid pGST·NHKI-CHKII, which encodes the chimeric
protein GST·NICII.
PCR-based Mutagenesis of Hexokinase
Constructs--
Site-directed mutations were generated by two separate
PCR reactions. The primers, templates, and PCR conditions used for the
construction of the hexokinase mutants are illustrated in Table
I. The PCR products were isolated by gel
electrophoresis. Equal amounts (approximately 100 ng) of the two
purified PCR products for each construct were mixed, denatured,
annealed, and used as a template for a third PCR reaction with the
outer primers, as described. By using these two primers, it was
possible to selectively amplify a template that consisted of annealed
PCR products 1 and 2 with the desired mutation. In the case of
pGST·HKII-S155A, the final PCR product was digested with
BamHI and SalI and was used to replace the
wild-type BamHI-SalI restriction fragment in the pGST·HKII plasmid. For pGST·HKII-S603A, the third PCR product was
digested with XhoI and ClaI and was used to
replace the corresponding wild-type fragment of pGST·HKII. In the
case of pGST·NHKI-D209A and pGST·NHKI-S155A, the final PCR products
were digested with SmaI and SalI and used to
replace the corresponding wild-type fragment of pGST·N-HKI.
pGST·NHKI-D209A and pGST·NHKI-S155A were digested with
BssHII and NcoI, and the smaller fragment was
used to replace the corresponding fragment of pGST·HKII to generate the plasmids pGST·NICII-D209A and pGST·NICII-S155A. These plasmids encode the chimeric proteins GST·NICII-D209A and GST·NICII-S155A. The PCR fragments inserted into the wild-type plasmids were validated by DNA sequencing.
Expression, Purification, and Activity Measurements of Hexokinase
Proteins--
The expression and purification of GST·HK fusion
proteins were carried out as described previously (7). Kinetic
parameters were determined by a glucose phosphorylation assay that uses
tracer [U-14C]glucose as substrate, as described
previously (7). The Km values for glucose and ATP
were determined by measuring the phosphorylation of
[U-14C]glucose as a function of added glucose (0.1-3
mM) and ATP (0.2-10 mM). The
KiG6P values were determined, with G6P
ranging from 0 to 10 mM, at a glucose concentration of 0.1 mM and an ATP concentration of 10 mM.
Analysis of Data--
A rate law for hexokinases was taken as
the model of the enzyme mechanism for the analysis of the kinetic data
(19). Garfinkel et al. derived this formula according to the
data of Grossbard and Schimke (20) and assumed a random order ternary
complex mechanism and assumed that G6P is a competitive inhibitor of
ATP and a noncompetitive inhibitor of glucose. A sensitive and precise tracer technique allowed us to measure small amounts of enzyme activity
at low substrate and high inhibitor concentrations (21). To use the
glucose tracer data directly in the determination of the kinetic
parameters, we modified the hexokinase rate law by dividing both sides
of the equation by the concentration of glucose to correct for the
specific activity of the tracer. The modified equation is as
follows.
Global Analysis--
Kinetic data were analyzed using a
nonlinear least squares curve-fitting program called Globals
UnlimitedTM (Department of Physics, University of Illinois,
Urbana, IL). This global analysis program was originally developed for
the analysis of multiple sets of fluorescence spectroscopic data (22, 23) and has been used to analyze other complex systems, such as coupled
glucose transport and phosphorylation (24). The practical aspects of
this approach have been reviewed (25). Global analysis was used to
generate nonlinear least square fits to the kinetic parameters of the
HKII enzymes based on the hexokinase rate law (see above). The kinetic
parameters (i.e. KmGlc,
KmATP, and
KiG6P) were determined by analyzing the
measured activities as a function of changes in each of the substrate
and inhibitor concentrations.
The kinetic parameters of HKII, its two halves (N-HKII and
C-HKII), and the D209A and D657A mutant enzymes are shown in Table II. In general, these are similar to the
apparent values obtained previously by simple regression analysis (7).
The exception is the KiG6P, which is
10-fold lower on average, because the current method of analysis
allowed for extrapolation to very low levels of the competitive ligand
ATP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Primers, templates, and PCR conditions used in the construction of
hexokinase mutants
In this equation, [Glc], [ATP], and [G6P] refer to the
concentrations of Glc, ATP, and G6P, respectively, and
KmGlc, KmATP, and
KiG6P are the respective kinetic constants for glucose, ATP, and G6P. Vmax is the
maximum velocity, and v is the velocity of glucose phosphorylation.
(Eq. 1)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Comparison of the kinetic values of various enzymes with HKII
The D209A mutation in HKII results in complete inactivation of the N-half of HKII (7), so the activity of the D209A enzyme is entirely contributed by its C-half. As noted with C-HKII, this mutant enzyme has a 20-30-fold higher KiG6P than HKII and N-HKII. Inactivation of the C-half of HKII (D657A) results in an enzyme with a KiG6P similar to that of N-HKII. However, neither the S155A mutation, which also inactivates the N-half of the enzyme, nor the S603A mutation affect the KiG6P of the intact enzyme (Table II). The KiG6P values for HKII, S155A, and S603A are between 39 and 45 µM, which is in agreement with results reported by Tsai and Wilson (8). These data suggest that S155A and D209A have different roles in HKII, particularly with respect to the regulation of the catalytic activity of the C-half by G6P. Previous studies have shown that the D209A mutation in mammalian hexokinases results in a loss of glucose binding and catalytic activity, whereas the S155A mutation maintains glucose binding but reduces the Vmax (5, 7, 17, 18). Therefore, the N-half of the D209A enzyme may remain in an open conformation, because the glucose-Asp209 contact is thought to trigger a conformational change from an open to a closed form. In contrast, the N-half of the S155A enzyme would undergo the conformational change associated with glucose binding, because the glucose-Ser155 contact occurs after the conformational change. We therefore hypothesize that a conformational change of the N-half is required for inhibition of the C-half at relatively low G6P concentrations.
Analysis of the kinetic parameters of the HKII enzyme, and its two halves, supports the hypothesis that the N-half influences the regulation of the C-half by G6P in the intact enzyme. The KiG6P of the intact enzyme is almost identical to that of N-HKII, despite the fact that the KiG6P of C-HKII is ~30-fold higher than that of the N-half. Detailed analysis of the G6P regulation of C-HKII in the intact enzyme is, however, complicated by the fact that both halves of HKII are active and both are sensitive to inhibition by G6P. We therefore constructed a chimeric enzyme in which C-HKII is the only catalytically active component and tested it for G6P inhibition.
The N-half of HKI is capable of binding glucose and G6P (1, 6), but it is catalytically inactive when expressed as a discrete protein and in the context of the intact HKI enzyme (Refs. 3-5 and data not shown). A chimeric protein consisting of the N-half of HKI and the C-half of HKII (NICII) allowed us to test whether glucose binding to the N-half, per se, is involved in the regulation of the C-half by G6P. In this chimeric enzyme the catalytic activity is entirely due to C-HKII, which has a significantly higher KiG6P than either HKI or HKII. The chimeric enzyme has KmGlc and KmATP values similar to those of C-HKII; however, the KiG6P is significantly lower than that of C-HKII (Tables II and III). These results indicate that the presence of the N-half of HKI causes a significant reduction in the KiG6P of the C-half of HKII in the context of a 100-kDa enzyme.
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We then asked whether the D209A or S155A mutations of this chimeric enzyme have an effect on the KiG6P similar to that seen when the same mutations are made in HKII. The D209A mutation was made in the N-half of NICII chimeric enzyme. This resulted in a significant increase in the KiG6P (Table III). The NICII enzyme, with the S155A mutation in the N-half, was then constructed. This mutation, which does not decrease glucose binding in other hexokinases, did not affect the KiG6P of this chimeric enzyme (Table III).
In the NICII chimeric enzyme, the two halves are not independent entities, and the presence of an N-half that is capable of binding glucose causes the C-half to be regulated at lower concentrations of G6P. Therefore, an allosteric interaction that affects the function of the intact enzyme appears to occur between the two halves of HKII. This interaction may change the structure of the C-half, causing a reduction in the KiG6P of the C-half of HKII. Alternatively, G6P bound to the N-half may result in a conformation that directly inhibits the activity of the C-half in the intact enzyme. The available data do not allow us to distinguish between these possibilities.
Regulation of the function of the C-half of HKII, by binding events that occur in the N-half, has precedent based on observations of HKI. A functional interaction between the two halves of HKI is determined by the binding of glucose, G6P, or Pi. For example, co-operative binding of glucose to N- and C-half sites in HKI was noted (26), an observation not expected for a monomeric enzyme, particularly in view of the fact that the N-half has no catalytic activity (3-5). The binding of either G6P or Pi results in a loss of cooperative binding of glucose with a corresponding change in the conformation of HKI (26). Also, the binding constant for G6P is substantially reduced in the absence of glucose (26).
Crystal structures of HKI complexed with glucose and G6P (13, 14, 16) or with glucose and Pi (15) were recently reported. These structures provide direct evidence, as first noted in the yeast HK (9-12), of transitions between open and closed conformations around the glucose binding cleft. The critical role of amino acids Asp209 and Asp657 in glucose binding was confirmed (13, 14, 16). Finally, a mutation of Arg801, which could provide a salt link between the two halves of HKI (15), significantly reduces the relief of G6P inhibition by Pi (27). Thus, the crystal structures of glucose and Pi bound to HKI support the concept of a functional interaction between the two halves of HKs and suggest that it is dependent upon bound glucose.
The characteristics of HKII described in this paper may relate to the
important role this enzyme plays in insulin-mediated glucose clearance
of skeletal muscle, cardiac muscle, and adipose tissue. The sequential
transport and phosphorylation of glucose by mammalian cells ensures
that the subsequent metabolic products are retained within the cell. In
the basal state, the intracellular glucose concentration is typically
low in muscle (28-31) and in fat (32, 33) but rises as the glucose
flux increases, either in response to increased extracellular glucose
or as a consequence of insulin stimulation. Recent in vivo
studies, using a variety of techniques, suggest that glucose
phosphorylation may be defective in individuals with
non-insulin-dependent diabetes mellitus (NIDDM). For
example, when positron emission tomography is used to measure 2-deoxy-[218F]fluoro-D-glucose transport
and phosphorylation, insulin infusion increases the rate constant for
phosphorylation in normal weight and obese controls but not in NIDDM
subjects (34). Bonadonna et al. (35) investigated
nondiabetic controls and patients with NIDDM using forearm balance
techniques during euglycemic glucose clamps. They found that glucose
transport is decreased about 40% but that combined transport and
phosphorylation is decreased 85% in patients with NIDDM. Also, in
31P NMR studies performed during
hyperglycemic-hyperinsulinemic clamps, the muscle content of G6P rises
with insulin infusion in controls but fails to increase with insulin
infusion in NIDDM subjects (36). We conclude that these patients have
defective glucose transport/phosphorylation. Finally, results from
muscle biopsies in persons with NIDDM from three distinct ethnic
backgrounds also led to the suggestion that the regulation of the HKII
gene is abnormal in this disease (37).
These observations are interesting with regard to the data reported in
this paper. G6P levels rise as glucose utilization increases. In fact,
intracellular G6P levels increase from ~170 to ~1300
µM in perfused working rat hearts either in the presence of 10 mM glucose or after 30 min of insulin treatment (38). Glycogen stores in these hearts increase 5-6-fold. If HKII is a
rate-determining step in glucose metabolism and is inhibited by low
concentrations of G6P (KiG6P of 45 µM), why does glucose utilization increase when G6P
increases? We suggest that HKII plays a role in insulin-sensitive
peripheral tissues similar to the one glucokinase exerts in the liver.
Glucokinase, due to its insensitivity to physiological concentrations
of G6P, continues to phosphorylate glucose at high levels of G6P, and
this results in increased hepatic glucose utilization and storage.
Glucokinase, when expressed in muscle cells, has a similar effect on
glucose disposal (39). We hypothesize that HKII, because of the low affinity of the C-half for G6P, may serve the same purpose in insulin-sensitive, peripheral tissues. This could be accomplished by
the binding of the enzyme to mitochondria and/or by some other effector
that results in allosteric modification of the enzyme that effectively
uncouples the functional interaction of the N- and C-halves of the enzyme.
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ACKNOWLEDGEMENTS |
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We thank Steve Koch for technical assistance and Deborah C. Brown for assistance in the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK46867 and DK20593 to the Vanderbilt Diabetes Research and Training Center.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.
¶ To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, 707 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-7004; Fax: 615-322-7236; E-mail: daryl.granner{at}mcmail.vanderbilt.edu.
2 H. Ardehali, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HK, hexokinase; G6P, glucose 6-phosphate; GST, glutathione S-transferase; kbp, kilobase pairs; PCR, polymerase chain reaction; Glc, glucose; NIDDM, non-insulin-dependent diabetes mellitus; C-half, C-terminal half; N-half, N-terminal half.
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