©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Active Site Residues of Human Brain Hexokinase as Studied by Site-specific Mutagenesis (*)

Chenbo Zeng , Herbert J. Fromm (§)

From the (1) Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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). 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 Kfor glucose but no change in either the Kfor 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 Kvalues for ATP and glucose changed only slightly. No change was observed in the Kvalue 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.


INTRODUCTION

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 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).

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) on glucose-6-P-inhibited mini-hexokinase.

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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 Kvalue for ATP increased 2-fold, and the Kvalue for glucose decreased 4.6-fold. Little change was observed for the Kvalues 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 Kvalues for glucose compared with wild-type mini-hexokinase; however, no alteration was observed on the Kfor 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.




DISCUSSION

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 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 Kfor 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.

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 on hexokinase I is in some way associated with the N-terminal half of the enzyme.

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 - 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 Kvalue for ATP increased 2-fold, whereas the Kvalue for glucose decreased 4.6-fold. No change was observed for the Kvalue 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.

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 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 Kvalues for glucose, but no change in either the Kfor 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



FOOTNOTES

*
This research was supported in part by National Institutes of Health Grant NS 10546, a grant from the United State Public Health Service, and National Science Foundation Grant MCB-9218763. This is Journal Paper J-16213 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project 2575. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Biochemistry and Biophysics, 1210 Molecular Biology Bldg., Iowa State University, Ames, IA 50011.

The abbreviations used are: DTT, DL-dithiothreitol; IPTG, isopropyl-1-thio--D-galactopyranoside; 1,5-An-G6P, 1,5-anhydroglucitol 6-phosphate; DNase I, deoxyribonuclease I; HPLC, high pressure liquid chromatography.


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