©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human -Cell Glucokinase
DUAL ROLE OF SER-151 IN CATALYSIS AND HEXOSE AFFINITY (*)

Liang Zhong Xu (3), Robert W. Harrison (1), Irene T. Weber (1), Simon J. Pilkis (2)(§)

From the (1) Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the (2) Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455, and the (3) Department of Physiology and Biophysics, Health Science Center, SUNY at Stony Brook, Stony Brook, New York 11794

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Glucokinase is distinguished from yeast hexokinase and low Kmammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Kfor glucose by factors of 2-, 26-, and 40-fold, respectively, decreased V, abolished cooperativity for glucose, and also decreased Kfor mannose and fructose. The Ser-151 mutants had hexose Kvalues similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Kvalues for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Kfor ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.


INTRODUCTION

Glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is expressed in pancreatic -cells and liver and is the first rate-limiting reaction in glycolysis in these tissues (1, 2, 3) . Glucokinase (hexokinase IV) is a member of the hexokinase family of enzymes but is distinguished from the other members of the family by its lower affinity for glucose ( K= 6.0 mM compared with 20-170 µM for the low Khexokinases), its molecular size (50 versus 100 kDa for hexokinase), and its cooperative behavior with respect to glucose (1, 2, 3, 4) . Recently, the sugar specificity of human -cell glucokinase has been studied by kinetic analysis, fluorescence techniques, and molecular modeling (5) . The enzyme exhibits sigmoid behavior with glucose and 2-deoxyglucose as substrates but not with mannose and fructose. A molecular model of human -cell glucokinase predicts that the carbonyl group of Ser-151 interacts with the 2-OH of mannose and fructose while Glu-256 interacts with the 2-OH of the glucose, which suggests that Ser-151 may be involved in the enzyme's cooperative behavior (5) .

The determinants of sugar specificity and cooperative behavior of human -cell glucokinase have also been studied by mutating several putative active site residues (6) , which are conserved in the NH- and COOH-terminal halves of mammalian hexokinase (N-HK() and C-HK, respectively), glucokinase, and yeast hexokinase (7) . The conserved putative glucose binding residues in human -cell glucokinase are Ser-151, Asn-204, Asp-205, Asn-231, Glu-256, and Glu-290 (8, 9, 10) . Asp-205 was predicted, by analogy with yeast hexokinase, to be a base catalyst, and consistent with that hypothesis, mutation of Asp-205 to Ala decreased the kof the enzyme by a factor of at least 1000 (9) . Asn-204, Gly-256, and Glu-290 were predicted from molecular modeling to interact with the 3-OH, 4-OH, 2-OH, and 1-OH groups of glucose (5, 10) . Mutation of these residues resulted in enzymes with decreased values of kand increased Kvalues for glucose, mannose, and 2-deoxyglucose (6) . These results support the prediction of the model that these residues interact with hexoses (5) . One conserved putative hexose binding residue of glucokinase that has not yet been studied by site-directed mutagenesis is Ser-151. In addition, based on the molecular model of glucokinase (10) , Asn-166 has been postulated to constitute one of the nonconserved residues whose corresponding residue in yeast hexokinase may be important for ATP binding and the glucose-induced conformational change (11) . It was the object of this study to examine whether Ser-151 and Asn-166 are involved in hexose binding and/or catalysis. It is demonstrated here that point site mutations of Ser-151 but not Asn-166 convert the high Kglucokinase to a lower Kenzyme similar, in some of its properties, to yeast hexokinase and the low Kmammalian hexokinases.


EXPERIMENTAL PROCEDURES

Materials

Q-Sepharose was from Pharmacia Biotech Inc. Glu-6-P dehydrogenase, Lactate dehydrogenase, and pyruvate kinase were from Boehringer Mannheim. Glucosamine, N-acetylglucosamine, mannose, fructose, and all other sugars were from Sigma. The full-length cDNA for human kidney hexokinase I was a kind gift from Dr. Graeme Bell (Howard Hughes Medical Institute, University of Chicago). Antibody to mouse brain hexokinase I was a kind gift from Dr. John Wilson (Dept. of Biochemistry, Michigan State University).

Construction of pET Expression Plasmids for Human -Cell Glucokinase

A 2.6-kilobase human pancreatic -cell glucokinase cDNA clone, ph-GK-20, was used to generate the construct pEhgk-WT (8) . An NdeI site was generated at the 5`-end using the polymerase chain reaction with an oligonucleotide that has a one-base pair mismatch (GGCTGGTGTGCATATGCTGGACGACAG). The insert in the pET3a expression construct included the protein coding region of the cDNA as well as the 3`-untranslated region.

Construction of pET3a HK-HK, N-HK, and C-HK Expression Plasmids

The expression plasmid pET3a HK-HK was constructed from an AlwNI- XbaI fragment of the human kidney hexokinase cDNA and two pairs of complementary oligonucleotide linkers. The linkers ( NdeI/ AlwNI, encoding the NH-terminal amino acids and translation initiation site) were phosphorylated, annealed, and ligated to the NdeI site of the pET3a vector and the AlwNI site of the cDNA. The 3`-linkers ( XbaI/ EcoRI), encoding the COOH-terminal phenylalanine and the termination site, were similarly treated and ligated to the XbaI site of the cDNA and to the EcoRI site of pET3a.

The pET3a C-HK (residues 456-920) expression plasmid was prepared by digestion of pET3a HK-HK with NdeI and NcoI, and the ends were filled in with Klenow and ligated together. The pET3a N-HK (residues 1-455) expression plasmid was prepared as follows. Polymerase chain reaction was used to amplify a fragment of pET3a HK-HK using the T7 forward primer and a synthetic primer, which contains a termination site right after the codon for Ala-455 and a XbaI site. Both the polymerase chain reaction fragment and pET3a vector were digested with XbaI. The pET3a vector was further treated with phosphatase and ligated to the XbaI-digested polymerase chain reaction fragment to construct the pET3a N-HK. The authenticity of all plasmid constructs and fidelity of the entire coding sequence of the cDNAs was determined by dideoxy chain termination sequencing with modified T7 DNA polymerase.

Construction of pET19b HK-HK, pET19b C-HK, and pET19b N-HK

pET19b HK-HK was prepared by digesting the construct pET3a HK and pET19b vector with NdeI and XbaI, followed by ligation. Both pET19b C-HK and pET19b N-HK were prepared in a similar manner.

Expression and Purification of HK-HK, C-HK, and N-HK

The expression of HK-HK (intact HK), C-HK, and N-HK were accomplished using the T7 RNA polymerase based system of Studier and Moffat (12) . Cells containing pET3a HK-HK and pET3a C-HK were induced at 22 °C for 24 h and harvested, and cell pellets were then lysed by freeze-thawing 3. The crude extracts containing HK-HK and C-HK were partially purified by 30% ammonium sulfate precipitation, and dialyzed against hexokinase buffer (5 mM potassium P, 1 mM EDTA, pH 8.0, and 10 mM glucose). The enzyme fractions were loaded on to a 100-ml DEAE-A50 ion exchange column and washed with hexokinase buffer containing 100 mM KCl. Hexokinase was eluted with a linear gradient of KCl (100-500 mM). The pooled fractions were concentrated by precipitation with 65% (NH)SO, dissolved in hexokinase buffer containing 100 mM KCl, and purified to homogeneity by elution from a Sephadex G-100 superfine column (1.5 90 cm). This purified preparation was free of bacterial hexokinase.

Purifications of pET19b HK-HK and pET19b N-HK were performed as follows. Cells harboring the expression plasmids were induced and harvested as described for pET3a HK-HK. Cell pellets were then lysed by freeze-thawing 3 except that a buffer containing 100 mM Tris, 100 mM KCl, pH 8.0, was used. The crude extracts were passed through a DEAE-Sephadex column equilibrated in the same buffer. The flow through was loaded onto a 10-ml Ni-Sepharose column, and the column was washed with 100 ml of buffer containing 10 mM imidazole, 100 mM Tris, pH 8.0, and then eluted with buffer containing 25 mM imidazole and 100 mM Tris, pH 8.0. The hexokinase fractions were precipitated with 65% ammonium sulfate, dissolved in 1 ml of hexokinase buffer, and purified to homogeneity on a Sephadex G-100 superfine column (1.5 90 cm) equilibrated with hexokinase buffer. The N-HK containing the His-tag sequence was induced and purified to near homogeneity in a similar manner. Western blots of both HK-HK and N-HK were performed using a polyclonal antibody against rat brain hexokinase.

Site-directed Mutagenesis of Human -Cell Glucokinase

Mutagenesis of human -cell glucokinase was carried out by using the method of Kunkel (13) . A NdeI- BamHI fragment of human pancreatic glucokinase was subcloned into pBluescript. A single-stranded uracil-containing DNA was prepared, annealed to a phosphorylated mutagenic oligonucleotide, and transformed into XL-blue cells. Mutant clones were identified by DNA sequencing. A NdeI- ClaI mutagenic fragment was subcloned into the pET3a GK vector. The entire NdeI- ClaI fragment was sequenced to confirm the authentic sequence of the pET3a GK expression vector. The double mutant (S151A and N166R) was prepared by subcloning the mutant fragment ( ClaI- SstI) of N166R into the S151A mutant expression vector digested by ClaI and SstI. Oligonucleotide primers were used to make mutations as follows with the mutated codon in the lower case: GK (wild type), 5`-CCCCTGGGCTTCACCTTCTCCTTTCCTGTGAGG-3`; S151C, 5`-CCCCTGGGCTTCACCTTCtgcTTTCCTGTGAGG-3`; S151A, 5`-CCCCTGGGCTTCACCTTCgccTTTCCTGTGAGG-3`; S151G, 5`-CCCCTGGGCTTCACCTTCggcTTTCC-TGTGAGG-3`; and N166R, 5`-GAAGACATCGATAAGGGCATCCTTCTCagaTGGACCAAGGGC-3`.

Bacterial Expression and Purification of Native and Mutant Forms of Human -Cell Glucokinase

Wild-type glucokinase and mutant enzymes were expressed in Escherichia coli BL21(DE3)-plys S using the phage T7 RNA polymerase-based system described by Studier and Moffat (12) , except that a lower induction temperature was used (9) . Similar yields of about 20 mg/liter were obtained for all of the mutants and for the wild-type enzyme. Cell extracts containing glucokinase were freeze-thawed three times to break the cells, and 100 µg/ml DNase A and 1 mg/ml lysozyme were added to the cell extract at 4 °C for 1 h. Glucokinase was first partially purified by precipitation with 40-65% (NH)SO. The pellet was dissolved in glucokinase buffer (50 mM potassium P, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride), dialyzed against the same buffer, and then loaded on to a 100-ml Q-Sepharose column. The column was washed with 2 liters of glucokinase buffer containing 150 mM KCl, and the enzyme was eluted with a linear gradient of 150-450 mM KCl. The fractions containing enzyme activity were pooled, precipitated with 70% (NH)SO, and dissolved in 1 ml of glucokinase buffer. Glucokinase was loaded on to a 100-ml G-100 Sephadex superfine column equilibrated in glucokinase buffer. The wild-type and mutant enzymes were eluted from the column with an apparent molecular mass of 50 kDa. Native human -cell glucokinase has a specific activity of about 80-120 units/mg, which is similar to that reported for the purified rat liver enzyme (14) . The various Ser-151 mutants were also purified to homogeneity by the above procedure.

Enzyme Assay and Kinetic Analysis

Glucokinase activity was assayed at 30 °C by measuring the increase in absorbance of NADPH at 340 nm in a coupled enzyme system, which employed Glu-6-P dehydrogenase as previously described (5, 9) . The Glu-6-P dehydrogenase assay was also used to determine the Kfor N-acetylglucosamine inhibition. When sugar specificity was studied, glucokinase activity was measured by monitoring the decrease in absorbance at 340 nm in an assay mixture containing 100 mM Tris-HCl (pH 7.5), 100 mM KCl, 20 mM MgCl, 0.6 mM phosphoenolpyruvate, 0.6 mM NADH, 1 mM ATP, 1.76 units/ml lactate dehydrogenase, and 1.4 units/ml pyruvate kinase. The Kfor Glu-6-P, with respect to glucose, was determined using the pyruvate kinase-lactate dehydrogenase coupling assay. The Vand Kfor substrates were obtained by the nonlinear least squares fitting program to fit a Michaelis-Menten equation or V = V S ( K+ S) for the Hill coefficient using Sigma Plot.

Modeling of Human -Cell Glucokinase Structure

A model of human -cell glucokinase was generated by analogy to the known crystal structure of the related yeast hexokinase (10, 11) followed by molecular mechanics minimization as described by Xu et al. (5) . The model and crystal structures were examined using the program FRODO (35) run on an Evans and Sutherland ESV 10 computer graphics workstation (5, 10) .

Determination of Circular Dichroism Spectra

All far UV spectra were collected on a Jasco 500 A in a 0.01-cm cell at 20 °C. Scans were collected at 1 nm/min with a time constant of 64 s, and all data are the average of three blank corrected samples. Samples were dialyzed against 50 mM potassium P, pH 7.5, 1 mM EDTA, and 1 mM dithiothreitol. Samples were centrifuged at 14,000 rpm for 10 min to remove any precipitated protein, and the Awas measured to scale the CD data to the same concentration. The CD data presented corresponds to samples with an Aof 1.0 in a 1-cm cell.

Determination of Intrinsic Fluorescence of -Cell Glucokinase

Experiments were performed in a buffer containing 50 mM potassium P, pH 7.5, 1 mM MgCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol with a multi-frequency phase fluorometer (ISS Inc.). The excitation wavelengths were 280 nm in a 10 10-mmsize cuvette (6, 15) . Glucose was removed from glucokinase prior to the experiment by a dialysis overnight against a buffer containing 50 mM potassium P, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol. The experiment was initiated by the addition of a final concentration of 1 or 100 mM hexose. The fluorescence spectra were recorded after addition of sugar and then analyzed with an IBM-PC microcomputer interfaced with the fluorometer to extract maximum fluorescence intensity of spectra at 312 nm. The relative fluorescence enhancement was obtained by dividing fluorescence enhancement by the initial intrinsic fluorescence.

Assay of ATPase Activity of Glucokinase

Reaction mixtures for following the ATPase activity of glucokinase contained 0-20 mM Mg-[-P]ATP (3 10cpm/umol), 5 mM excess MgCl, 50 mM Pipes, pH 7.0, and 20 µg of glucokinase in 200 µl. Inorganic phosphate release was performed by the method of Viola et al. (16) as previously described (5) .


RESULTS

Expression and Purification of Wild-type and Mutant Glucokinases

Native and mutant human -cell glucokinases were expressed in E. coli using the pET expression system essentially as previously described (5, 8, 9) . Glucokinase was purified 20-fold from extracts of E. coli by (NH)SOprecipitation (45-65%), Q-Sepharose chromatography, and gel filtration on a Sephadex G-100 superfine column as described under ``Experimental Procedures'' (20) . The Q-Sepharose column is the most important step for removing contaminating bacterial hexokinase, which was eluted with the 150 mM KCl, potassium P, pH 8.0, wash. Wild-type glucokinase and all five Ser-151 and Arg-166 mutants were purified to homogeneity by the criteria of SDS-PAGE (data not shown).

Circular Dichroism of the Mutants and Wild-type Enzyme

To ascertain whether the point site mutations caused large changes in secondary structure, the circular dichroism spectra of the mutants were compared with wild-type enzyme. There were no significant changes in the circular dichroism spectra of the Ser-151 mutants (Fig. 1), which indicates that there was no gross change in their secondary structure. There was also no change in the circular dichroism of the Asn-166 mutants (data not shown).


Figure 1: CD spectra of wild-type glucokinase and various Ser-151 mutants. The circular dichroism data were blank corrected and scaled for concentration.



Effect of Mutation of Ser-151 and Asn-166 on Glucokinase Phosphorylation of Glucose

Two distinctive features of mammalian glucokinase are its high Kfor glucose and cooperative behavior with respect to glucose (1, 2, 3, 4) . The effect of various Ser-151 mutations is shown in . Mutation of Ser-151 to cysteine decreased the Kfor glucose to one-third, while kdecreased to about one-tenth the wild-type value. Mutation of Ser-151 to Ala decreased the Kfor glucose by a factor of nearly 20, while kdecreased to one-tenth the wild-type value. The S151G mutant had a Kabout 1/40th the wild-type value, while kdecreased by 2 orders of magnitude. The Kvalues for ATP were increased about 5-fold for all Ser-151 mutants. The sigmoid behavior of glucokinase with regard to glucose was abolished for the S151A and S151G mutants.

Mutation of Asn-166 to Arg lowered the Kvalues for glucose and for ATP to one-third the wild-type enzyme value, while Vand the Hill coefficient were not affected. Therefore, the effect of mutating both Ser-151 and Asn-166 was tested. The double mutant, N166R/S151A, exhibited a Kfor glucose about 60% lower than that for the S151A mutant, but the Vwas also decreased by a factor of 3. Like the S151A mutant, the double mutant had a Hill coefficient of 1.0 and a higher Kfor ATP.

Effect of Mutation of Ser-151 and Asn-166 on Sugar Specificity of Glucokinase

The sugar specificity of human -cell glucokinase has been studied (5) . Mannose, 2-deoxyglucose, and fructose are substrates of the enzyme, and it was of interest to determine whether the Ser-151 mutants also had enhanced affinities for these substrates. As shown in , for the S151C mutant, the Kvalues for mannose and fructose were similar to that for the wild-type enzyme, but the kvalues relative to glucose were decreased. The S151A mutant exhibited decreased Kvalues for mannose, 2-deoxyglucose, and fructose by factors of about 20, 10, and 4, respectively, compared to the wild-type enzyme. The Vvalues decreased by factors of 20 for mannose and 2-deoxyglucose and by about 5-fold for fructose compared to wild-type enzyme. The Ser-151 mutants had similar catalytic efficiencies for glucose and mannose, except for S151C, which phosphorylated mannose less efficiently than glucose. S151A was similar to the wild-type enzyme in catalytic efficiency for fructose, but the Cys and Ala mutants were at least 1 order of magnitude less efficient at phosphorylating fructose compared to the wild-type enzyme.

N166R exhibited a 2-fold decrease in Kfor mannose compared to the wild-type enzyme, resulting in nearly 2-fold greater catalytic efficiency. For fructose, this mutant showed an increase in Vand decreased K, resulting in nearly 10-fold greater catalytic efficiency compared to the wild-type enzyme. The double mutant had greatly enhanced affinities for mannose and fructose.

Effect of Ser-151 Mutations on Glucose-induced Changes in Intrinsic Fluorescence

Rat liver glucokinase exhibits a glucose-induced change in intrinsic fluorescence that has been postulated to reflect the presence of two different conformations of the enzyme (15, 17) . In the crystal structures, yeast hexokinase is observed in an open conformation in the presence of a glucose analog inhibitor and in a closed conformation in the complex with glucose (18) . Human -cell glucokinase has been predicted to undergo a similar conformational change on the binding of glucose (5, 6, 10, 18) . For the wild-type enzyme, 100 mM glucose caused a 31.1% increase in intrinsic fluorescence, while 1 mM glucose had no significant effect. The S151C mutant exhibited a small but significant increase (4.2%) in intrinsic fluorescence at 1 mM glucose and the same change at 100 mM glucose as seen with the wild-type enzyme. However, the S151A mutant exhibited a 41.5% change in intrinsic fluorescence at 1 mM and a 54.8% increase at 100 mM glucose. The results show that in the case of the S151A mutant, the glucose dependence of changes in intrinsic fluorescence parallels the changes in hexose affinity. The N166R mutant exhibited a reduced glucose-induced change in intrinsic fluorescence (4% at 1 mM glucose and 10% at 100 mM glucose).

Effect of Mutations on Inhibition of Glucokinase by N-Acetylglucosamine and Glu-6-P

I shows that the Kvalues for the competitive inhibitor N-acetylglucosamine (5, 6, 18) for all of the Ser-151 mutants were similar to that of wild-type enzyme, even though the S151A and S151G mutants exhibited dramatically decreased Kvalues for glucose. The Kvalues for Glu-6-P of all Ser-151 mutants were also similar to that for the wild-type enzyme, with small decreases for S151C and S151A (I). The results demonstrate that mutation of Ser-151 has little effect on the enzyme's inhibition by Glu-6-P and N-acetylglucosamine. The N166R mutant and the double mutant showed increased affinity for N-acetylglucosamine by about 2-3-fold but no change in Kfor Glu-6-P.

Effects of the Ser-151 Mutations on ATPase Activity of Glucokinase

Yeast hexokinase exhibits low ATPase activity (19, 20, 21) , which is inhibited by phosphorylation of Ser-158, a side reaction product of ATP hydrolysis (20, 21) . Glucokinase also has a low ATPase activity, and Ser-151 corresponds to Ser-158 of yeast hexokinase (10) . Mutation of Ser-151 to Cys or Ala abolished the ATPase activity as shown in Fig. 2. For comparison, the N204Q mutant showed easily detectable ATPase activity, even though Vdecreased by 10-fold compared to the wild-type enzyme (6) . The specific activities ( V) of the kinase reaction were similar for the N204Q (6 units/mg), S151C (6 units/mg), and S151A (6 units/mg) mutants. No ATPase activity was measured with the Ser-151 mutants, even when three times more mutant enzyme protein than wild-type enzyme was used. These results suggest that Ser-151 is directly involved in the hydrolysis of ATP.


Figure 2: ATPase activity of human -cell glucokinase and various mutants. Glucokinase ATPase activity was measured by following the production of P from [-P]ATP as described under ``Experimental Procedures.'' Eight different ATP concentrations in the range of 0-20 mM were used.



Kinetic Analysis of HK-HK, C-HK, and N-HK

Mutation of the active site residue Ser-151 converts the high Kglucokinase to a low Kenzyme. To compare the glucokinase Ser-151 mutant forms with low Khexokinases of the same molecular size, the cDNAs corresponding to the NH- and COOH-terminal halves of hexokinase I were engineered into expression vectors. The BL 21(DE3)pLysS strains harboring pET3a HK-HK, C-HK, and N-HK were induced at 37 and 22 °C by isopropyl-1-thio--D-galactopyranoside. The cells harboring the pET3a vector were used as a control. The yield of active soluble protein at 37 °C was low (0.3 mg/liter). HK-HK was induced rapidly at this temperature and reached a plateau at 5 h and decreased afterward. C-HK was induced more quickly and reached a plateau at 10 h. In contrast, N-HK was induced very well as judged by [S]Met incorporation (data not shown), but hexokinase activity was insignificant, suggesting that N-HK is not active. In contrast to the low yield of active soluble enzyme at 37 °C, the yield of soluble, active C-HK was 10-fold higher at 22 °C (3 mg/liter). HK-HK was also induced to a higher level at this temperature and reached a plateau of 2.5 mg/liter at 20 h. As was the case at 37 °C, N-HK was induced very well at 22 °C (data not shown), but again the hexokinase activity was the same as that of the pET3a vector alone.

The results clearly show that the yields of active hexokinase forms were better at 22 °C than 37 °C, probably because the protein folds more readily at 22 °C. However, N-HK was not active even though different strains and different temperatures were tested (data not shown). Both HK-HK and C-HK were induced (2-3 mg/liters) but not nearly to the same extent as glucokinase (20 mg/liters). HK-HK and C-HK were partially purified by 15-30% ammonium sulfate fractionation and DEAE-A50 ion exchange chromatography with a linear gradient of KCl (100-500 mM). The partially purified enzymes were free of bacterial hexokinase. The kinetic properties of both HK-HK and C-HK were characterized (). For HK-HK, the Kfor glucose was 28.2 µM, the Kfor ATP was 0.47 mM, and the Kfor Glu-6-P was 70 µM. Interestingly, C-HK retained essentially the same kinetic constants as the intact hexokinase (HK-HK) (). The results suggest that the Glu-6-P inhibition site and catalytic site are located in the C-HK domain, consistent with the notion that Glu-6-P binds at the catalytic site and/or its binding site overlaps with it.

To confirm that the N-HK is not active, it was necessary to undertake its purification. However, this form was recalcitrant to purification (data not shown). A His-tag system based on the T7 RNA polymerase expression system, in which the HK cDNA is fused with a His-tag leader sequence, was used to circumvent the problem. Comparison of the kinetic properties of HK-HK with the His-tag sequence with the native HK-HK revealed no major differences with respect to kinetic properties (). N-HK with the His-tag sequence was induced and purified but had no measurable activity. Western blots with a polyclonal antibody against brain hexokinase confirmed the identity of the purified N-HK and HK-HK (Fig. 3). The results provide definitive evidence that purified N-HK has no enzymatic activity.


Figure 3: A, SDS-PAGE of purified His-tag-HK-HK and His-tag-N-HK. About 10 µg of purified His-tag-HK-HK and His-tag-N-HK were subjected to SDS-PAGE. Lanes 1-3 are molecular marker, HK-HK, and N-HK, respectively. B, Western blot of purified His-tag-HK-HK and His-tag-N-HK. About 10 µg of purified His-tag-HK-HK and His-tag-N-HK were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blotted with nonfat milk and probed with polyclonal antibody against mouse brain hexokinase I. Lanes 4 and 5 are HK-HK and N-HK, respectively.




DISCUSSION

Mammalian low Khexokinases are not expressed in high yield in bacterial expression systems (28, 29, 30) in contrast to the lower molecular weight glucokinases (6, 8, 9) , even though NH- and COOH-terminal halves of hexokinase share extensive similarity with glucokinase (22, 23, 24, 25, 26) . Expression and characterization of the NH- and COOH-terminal halves of hexokinase revealed that C-HK retained the same catalytic activity, with a high affinity for glucose and a marked inhibition by Glu-6-P, as intact native hexokinase I (27, 28, 29) . This is the first instance where the NH- and COOH-terminal halves of low Khexokinase have been purified. The NH-terminal form had no activity in its native form (pET3a vector) or as a fusion protein (pET19 vector), while the intact hexokinase was active in both instances. The results strongly suggest that the NH-terminal half of hexokinase I is not responsible for the enzyme's catalytic activity or sensitivity to Glu-6-P inhibition. It is possible the NH-terminal half may mediate hexokinase I interaction with the mitochondria or other subcellular structures (31, 32) . It is not known whether the NH-terminal halves of hexokinase II or III are also inactive. Hexokinase II has been postulated to have arisen as a result of gene duplication of a ``glucokinase''-like gene (23, 33) . If this is true, the gene duplication and tandem ligation of the two hexokinase halves per se may be responsible for the low affinity for glucose, or alternatively, subsequent mutations in the NH- or COOH-terminal halves may have resulted in an increase in glucose affinity. The former possibility seems unlikely in view of the findings with hexokinase I reported here. This question can be approached by characterization of the NH- and COOH-terminal halves of hexokinase II, and such work is in progress.

In contrast to the low Khexokinases, glucokinase has a low affinity for glucose ( K= 6 mM) and does not exhibit product inhibition by physiological concentrations of Glu-6-P ( K= 12 mM) (1, 2, 3, 4) (). Yeast hexokinase shares 33% identity with glucokinase but exhibits a high affinity for glucose ( K= 100 µM) and lack of inhibition by Glu-6-P ( K= 20 mM) (16) , even though all the putative glucose-binding residues are conserved in C-HK, N-HK, glucokinase, and yeast hexokinase (22, 23, 24) . Recently, we have shown that mutation of the active site residues, Asn-204, Glu-256, or Glu-290 to Ala in the human -cell glucokinase results in an increased Kfor glucose and a decreased kwith loss of sigmoid behavior with glucose as substrate. The cooperative behavior of glucokinase may thus be mediated by interactions of other nonconserved regions of the protein with the highly conserved active site glucose-binding residues (6) . Analysis of the crystal structure of yeast hexokinase led to the selection of Asn-166 as one such nonconserved residue. Its location, and that of Ser-151, in the glucokinase structure is shown in Fig. 4 , which depicts a stereo view of the open form of glucokinase based on the crystal structure of yeast hexokinase (5, 10, 11) . Both residues are located in the active site cleft in the smaller of the two domains. In the open conformation of yeast hexokinase, the residue corresponding to glucokinase 166 is Arg-173, and Arg-173 was predicted to form an interdomain hydrogen bond interaction with Gln-298 (11) . The Arg-173 to Gln-298 interaction spans the sugar-binding cleft and possibly may be involved in the conformational change induced by substrate binding. In addition, Arg-173 provides a positively charged residue near the expected ATP-binding site. Mutation of Asn-166 to Arg in glucokinase would restore this positive charge, which would be predicted to promote the binding of ATP and therefore the phosphorylation of sugar. These predictions were born out by kinetic analysis of the N166R mutant, which revealed an enhanced affinity for both ATP and glucose. However, this single point site mutation did not affect cooperativity or convert the high Kenzyme to a low Khexokinase-like form, although its apparent affinity for mannose and fructose was also increased.


Figure 4: Stereo view of the open conformation of human glucokinase. The mutated residues Ser-151 and Asn-166 are located close to the bound glucose in the model structure of human glucokinase. The carbon backbone of the glucokinase model is shown ( continuous line) with the side chain atoms of Ser-151, Asn-166, and glucose ( ball and stick representation).



This report strongly suggests that Ser-151 plays a critical role in determining the hexose affinity and cooperativity, as well as catalysis, of the enzyme. The residue equivalent to Ser-151 in yeast hexokinase is Ser-158, which is phosphorylated during the ATPase reaction. Also, molecular modeling of glucokinase predicted that the carbonyl oxygen of Ser-151 would form a hydrogen bond interaction with the 2-hydroxyl group of mannose (5) . This analysis suggested that 1) Ser-151 was located near the phosphate of ATP and 2) Ser-151 was implicated in the cooperative effect of glucose (5) . When the crystal structures of yeast hexokinase in the open and closed conformations are compared, differences are seen in the position and interactions of Ser-158. In the open conformation of yeast hexokinase, the hydroxyl side chain of Ser-158 was within hydrogen bonding distance of the amino acid side chains of Asp-86 and Lys-176. However, after the conformational change to the closed form, Ser-158 moved closer to glucose so that its hydroxyl group still interacted with Asp-86 but no longer interacted with Lys-176, and the carbonyl oxygen of Ser-158 formed a hydrogen bond interaction with the 3-hydroxyl group of glucose. Apparently, only after the conformation change does Ser-158 become a glucose-binding residue. We propose that a similar conformational change occurs in glucokinase. This suggests an explanation for the lower Kaccompanied by a lower reaction rate for the Ser-151 mutants. In each of the mutations of Ser-151 to Cys, Ala, and Gly, the hydroxyl side chain of Ser and any interactions with the neighboring Asp-78 and Lys-169 have been eliminated. This may facilitate the conformation change and movement of residue 151 to the position in which the carbonyl oxygen can interact with the 3-hydroxyl group of glucose (Fig. 5), thus increasing the apparent affinity for glucose. Alternatively, the non-productive binding of hexoses or ATP may be reduced by eliminating potential interactions with residue 151. The glucose-induced change in conformation of residue 151 may be more effective when the Ser hydroxyl group and its interactions with as yet undefined neighboring residues are eliminated.


Figure 5: Top panel, schematic representation of the interactions of Ser-151 in the open conformation of human glucokinase as predicted by analogy with the crystal structure of yeast hexokinase B. Potential hydrogen bond interactions are indicated by dashed lines. Ser-151 interacts with the side chains of Asp-78 and Lys-169. Lower panel, schematic representation of the interactions of Ser-151 in the closed conformation of human glucokinase as predicted by analogy with the crystal structure of yeast hexokinase A with glucose. Potential hydrogen bond interactions are indicated by dashed lines. Ser-151 interacts with the side chain of Asp-78 and the 3-OH of glucose.



However, the loss of hydrogen bond interactions among the residues forming the glucose binding site is expected to destabilize the enzyme structure and result in a reduced reaction rate. For example, mutation of Asn-204, Glu-256, and Glu-290 to Ala in the human -cell glucokinase resulted in increased Kvalues for glucose, decreased kvalues, and diminished conformational changes upon addition of 100 mM glucose, as measured by the change in intrinsic fluorescence (6) . In the case of Ser-151 mutations, the greatest decrease in catalysis was observed for the S151G mutant, which is probably less stable due to the flexible glycine. S151A is the most effective mutation, since it apparently retains some structural stability due to the short alanine side chain and undergoes the glucose-induced conformational change at low glucose concentration, as shown by fluorescence changes. The residue in hexokinase I corresponding to Ser-151 in glucokinase has been mutated with loss of enzyme activity (33, 34) consistent with the results reported here, but the mutated enzyme was not purified nor was its kinetic properties characterized in detail.

N-Acetylglucosamine is an effective competitive inhibitor of glucokinase with respect to glucose. The Kvalues for N-acetylglucosamine were unchanged for the various Ser-151 mutants even though Kfor glucose was dramatically decreased to values near that for low Khexokinase and yeast hexokinase. These results suggest that N-acetylglucosamine is not contacted by Ser-151. Consistent with this hypothesis, Xu et al. (5) modeled N-acetylglucosamine binding to the open conformation of the enzyme and predicted that Ser-151 was not involved in binding the inhibitor. This report also confirms previous studies that the catalytic site of mammalian low Khexokinase with high affinity for glucose and Glu-6-P is in the COOH-terminal half of hexokinase, while N-HK is not active (27, 28, 29) . Although the S151A and S151G mutants have similar affinity for glucose as do C-HK and yeast hexokinase (), mutation of Ser-151 did not generate a sensitive Glu-6-P inhibitory site in glucokinase, suggesting that the Ser-151 mutants resemble yeast HK more than C-HK ().

ATPase activity is conserved in both yeast hexokinase and glucokinase (5, 19, 20, 21) . Mutation of Ser-151 to Cys or Ala reduced the ATPase activity below detectable levels, which suggests that the OH group of Ser-151 attacks the -phosphate of ATP. In the kinase reaction, the carboxylate group of Asp-205 is involved in transferring the -phosphate of ATP to the 6-OH group of glucose (9) . The Ser-151 mutations also resulted in about a 5-fold increase in the Kfor ATP and reduced the V. The results reported here suggest that Ser-151 is physically close to the base catalyst, Asp-205. In the yeast hexokinase closed form crystal structure, the side chains of the equivalent Ser-158 is 4.4 A from the side chain of the catalytic Asp-211.

In summary, this study has provided clues to explain glucokinase's low affinity for glucose. While mutation of most of the enzyme's putative glucose binding residues dramatically increased the Kfor glucose (6) , mutation of Ser-151 decreased the Kfor glucose into the range found in yeast hexokinase and low Khexokinases (), eliminating cooperative behavior and converting the high Kglucokinase to a low Kenzyme but with a compromised catalytic rate. This implies that it might not be possible to convert the high Kto a low Kenzyme by a single point mutation, with no change in k. This view is supported by results of the double mutation, N166R/S151A. However, as judged by changes in intrinsic fluorescence of S151A, the glucose-induced conformational change occurs at an even lower glucose concentration than for the wild-type enzyme, suggesting that the lower kis not due to decreased ability of the enzyme to undergo the glucose-induced conformational change but rather is due to distortions in the active site or related protein regions that prevent the efficient transfer of the -phosphate of ATP to the 6-OH group. However, it also leaves open the possibility that a low K``glucokinase'' with no change in turnover number can be generated by site-directed mutagenesis of Ser-151 and other nonconserved residues in glucokinase. Such work is in progress.

  
Table: Effect of mutation of Ser-151 on -cell glucokinase catalysis and affinity for glucose

Glucokinase activity was assayed at 30 °C by measuring the change in absorbance of NADPH at 340 nM as described under ``Experimental Procedures.'' Each data point represents the average ± S.E. of triplicate independent curve fits. Each curve was obtained from triplicate measurements at each of 9-12 different glucose concentrations. The range of glucose concentrations for the wild-type and mutant enzymes used depended on the Kof the enzyme. For the wild-type enzyme, glucose concentrations in the range of 0.1-100 mM were used. For the S151G mutant, glucose concentrations in the range of 0.05-1 mM were used. V, K, and Hill coefficient were determined by the non-linear least squares routine using Sigma plot.


  
Table: Effect of mutation of Ser-151 and Asn-166 on the sugar specificity of -cell glucokinase

Glucokinase activity was measured by monitoring the decrease in absorbance at 340 nm using the lactate dehydrogenase-pyruvate kinase-coupled assay as described under ``Experimental Procedures.'' The concentration of sugar analogs used depended on the Kof the enzyme and was between 0.05 and 600 mM. V, Kwere determined by non-linear least squares routines using Sigma plot. ND designates not determined.


  
Table: Effect of mutation of Ser-151 on competitive inhibition of -cell glucokinase by N-acetylglucosamine and Glu-6-P

Glucokinase activity was measured by using the glucose-6-phosphate dehydrogenase coupling assay, with varying concentrations of N-acetylglucosamine at different concentrations of glucose. For Glu-6-P inhibition, glucokinase activity was measured by using the pyruvate kinase-lactate dehydrogenase assay, with varying concentrations of Glu-6-P at different concentrations of glucose. Vand apparent Kwere determined by using non-linear least squares methods to fit the Michaelis-Menten equation using Sigma plot. Kwas obtained by Dixon plot (l/v versus I). K/ Krepresents the ratio of the Kfor inhibition over the Kfor glucose. The data represent the averages of three separate determinations.


  
Table: Kinetic properties of hexokinase

Hexokinase activity was measured by using the glucose-6-phosphate dehydrogenase coupling assay, with varying concentrations of glucose with a range of 0.01-1 mM at 5 mM ATP or with varying concentrations of ATP with a range of 0.1-10 mM at 100 mM glucose. For Glu-6-P inhibition assay, hexokinase activity was measured by using pyruvate kinase-lactate dehydrogenase assay, with varying concentrations of Glu-6-P at three different concentrations of ATP (0, 0.5, and 1 mM). Vand apparent Kwere determined by using non-linear least squares methods to fit the Michaelis-Menten equation using Sigma plot. Kwas obtained by Dixon plot (l/v versus I). ND designates not determined. - designates no inhibition for the bacterial form. The data represent averages of three separate determinations of each kinetic parameter.


  
Table: Comparison of N-HK, C-HK, yeast hexokinase, and glucokinase

Data for yeast HK are from Ref. 16. All the other data are from Ref. 6 and this report. ND, not determined.



FOOTNOTES

*
This research was supported by National Institutes of Health Grant R01-DK46562 (to S. J. P.) and a grant from the American Diabetes Association (to I. T. W.). 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 correspondence should be addressed. Tel.: 612-625-6100; Fax: 612-625-2163.

The abbreviations used are: N-HK, the NH-terminal half of human kidney hexokinase I; C-HK, the COOH-terminal half of human kidney hexokinase I; HK-HK, human kidney hexokinase I; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; GK, glucokinase.


ACKNOWLEDGEMENTS

We thank Kristina Johnson for skillful typing of the manuscript.


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