The Roles of Glycine Residues in the ATP Binding Site of Human Brain Hexokinase*

Chenbo ZengDagger , Alexander E. Aleshin, Guanjun Chen§, Richard B. Honzatko, and Herbert J. Fromm

From the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutants of hexokinase I (Arg539 right-arrow Lys, Thr661 right-arrow Ala, Thr661 right-arrow Val, Gly534 right-arrow Ala, Gly679 right-arrow Ala, and Gly862 right-arrow 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 right-arrow Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 right-arrow Ala mutant exhibited a 4000-fold decrease in kcat and the Gly862 right-arrow 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 right-arrow Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 right-arrow Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 right-arrow Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 right-arrow Ala and Gly679 right-arrow Ala mutants. Gly862 right-arrow Ala and Gly534 right-arrow 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 right-arrow Ala, Arg539 right-arrow Lys, Thr661 right-arrow Ala, Thr661 right-arrow Val, Gly679 right-arrow Ala, and Gly862 right-arrow Ala mutants of brain hexokinase. The mutation Gly862 right-arrow 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 right-arrow 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta -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-beta -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 right-arrow 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 right-arrow Ala, 5'-CTCATTGTTGCGACCGGC-3' for Gly679 right-arrow Ala, 5'-GTGGACGCGACACTCTAC-3' for Gly862 right-arrow Ala, 5'-CAGTGGGCGCAATGATGACC-3' for Thr661 right-arrow Ala, 5'-CAGTGGGCGTCATGATGACC-3' for Thr661 right-arrow Val, 5'-ACCAATTTCAAAGTGCTG-3' for Arg539 right-arrow 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 beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow Ile mutant was relatively insoluble (20). Pi reverses Glu-6-P inhibition of the Gly679 right-arrow 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 right-arrow Ala and Gly534 right-arrow Ala mutants, respectively (Fig. 1).

                              
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Table I
A comparison of the kinetic data of the wild-type and the mutant forms of brain hexokinase


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Fig. 1.   Reversal of 1,5-anhydroglucitol-6-phosphate inhibited wild-type and mutant hexokinase enzymes by Pi. The concentrations of ATP are 0.25 mM (gray), 1 mM (black), and 4 mM (white) for the wild-type, Gly534 right-arrow Ala (G534A), Gly679 right-arrow Ala (G679A), Gly862 right-arrow Ala (G862A), Thr661 right-arrow Ala (T661A), Thr661 right-arrow Val (T661A), and Arg539 right-arrow Lys (R539K) enzymes, and 4 mM (gray), 8 mM (black), and 16 mM (white) for the Gly862 right-arrow Ala mutant. Pi concentration is 5 mM, and the concentration of 1,5-anhydroglucitol-6-phosphate is 100 µM. Reversal of 1,5-anhydroglucitol-6-phosphate inhibition (%) is defined as (A - B) × 100/B where A is activity in the presence of 1,5-anhydroglucitol-6-phosphate and Pi, and B is activity in the presence of 1,5-anhydroglucitol-6-phosphate.

The fluorescence emission spectra of the wild-type, Gly534 right-arrow Ala and Gly679 right-arrow Ala enzymes were identical, whereas the spectrum for the Gly862 right-arrow Ala mutant differed from that of the wild-type enzyme (Fig. 2). This difference is probably due to changes in the local environment of tryptophan residues. However, the CD spectra for the wild-type and all the mutant enzymes are identical (data not shown), suggesting no global conformational differences among these proteins.


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Fig. 2.   Fluorescence emission spectra for the wild-type and Gly862 right-arrow Ala mutant form of human brain hexokinase.

The Thr661 right-arrow Ala mutant showed a 4-fold increase in the Ki for 1,5-anhydroglucitol-6-phosphate. The Thr661 right-arrow Val mutant exhibited a 9-fold decrease in kcat relative to the wild-type enzyme. Other kinetic parameters for the two mutants were unaltered relative to those of the wild-type enzyme. The Arg539 right-arrow Lys mutant showed a 12-fold decrease in kcat, and little change in the Km for either ATP or glucose or the Ki for 1,5-anhydroglucitol-6-phosphate.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Arg539 putatively interacts with the polyphosphoryl portion of ATP and stabilizes the transition state (19, 20). The Arg539 right-arrow Lys mutant is 10-fold more active than the Arg539 right-arrow Ile mutant (20), suggesting the importance of the positive charge at position 539. However, as the Arg539 right-arrow 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 alpha - and beta -phosphoryl groups of ATP (19). The observed properties of the Lys539 mutant are consistent with that suggestion.

Thr661 is 10 Å away from the beta -phosphoryl group of ATP in our model, however, the gamma -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 right-arrow 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, Cbeta of Ala534 is 3.6 Å from an oxygen of the beta -phosphoryl group of ATP, but perhaps of greater significance is its 2.4 Å contact with backbone carbonyl 537 of an adjacent beta -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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Fig. 3.   Stereoview of the model for ATP in its complex with hexokinase I. Positions 534, 679, and 862 are presented in their mutated states as alanines with bold lines. ATP and glucose (GLU) are also drawn in bold lines, and the solid circle represents Mg2+.

The Gly534 right-arrow 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 right-arrow 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 (phi  = -111, psi  = -133 for Gly679; phi  = 70, psi  = 168 for Gly862). Of the two mutants, Gly862 right-arrow Ala has the conformation of highest energy. Although the Cbeta 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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).

    ACKNOWLEDGEMENT

We thank Laura Duck for excellent technical assistance.

    FOOTNOTES

* 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.

Dagger 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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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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