©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino Acid Conservation in Animal Glucokinases
IDENTIFICATION OF RESIDUES IMPLICATED IN THE INTERACTION WITH THE REGULATORY PROTEIN (*)

(Received for publication, October 10, 1995; and in revised form, December 21, 1995)

Maria Veiga-da-Cunha (1)(§)(¶) Stephane Courtois (1)(§)(**) Alain Michel (2) Eric Gosselain (1) Emile Van Schaftingen (1)(§§)

From the  (1)Laboratory of Physiological Chemistry, University of Louvain and the International Institute of Cellular and Molecular Pathology, B-1200 Brussels and the (2)Biological Chemistry Department, Faculty of Sciences, University of Mons-Hainaut, B-7000 Mons, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To delineate the regions of liver glucokinase that are involved in the binding of its regulatory protein and have therefore been conserved throughout evolution, we have cloned the cDNA of the Xenopus laevis enzyme. It contains an open reading frame of 1374 nucleotides and encodes a protein of 458 amino acids, which displays 78 and 79% overall identity to rat and human liver glucokinases, respectively. The conserved regions are predicted to be present mainly in the small domain and the hinge region of glucokinase, and the nonconserved regions in the large domain of the enzyme.

We constructed five mutants of Xenopus glucokinase by replacing sets of 2-5 glucokinase-specific residues with their counterparts in the C-terminal half of rat hexokinase I. The affinity for the regulatory protein was not markedly changed for mutants B, D, and E despite a decreased affinity for glucose in mutants B and D. Two other mutants (A and C) were 9- and 250-fold less sensitive to the rat regulator and 40- and 770-fold less sensitive to the Xenopus regulator, respectively, but presented a normal affinity for glucose. The double mutant (A-C) was completely insensitive to inhibition by the regulatory protein. A control mutant (F), obtained by replacing 3 residues that were not conserved in all glucokinases, had a normal affinity for glucose and for the regulatory protein. The property of glucokinase to be inhibited by palmitoyl-CoA was not affected by the mutations described. It is concluded that His-141 to Leu-144, which are located close to the tip of the small domain, as well as Glu-51 and Glu-52, which are present in the large domain of the enzyme close to the hinge region, or nearby residues participate in the binding of the regulatory protein.


INTRODUCTION

The hexokinase family consists of several evolutionarily related enzymes, all of which catalyze the phosphorylation of glucose. In this family, animal glucokinases (also called hexokinase D or IV) are characterized by a number of kinetic properties that are not shared with the other hexokinases: 1) a low affinity for glucose, 2) a sigmoidal saturation curve for this substrate, and 3) the property to be inhibited by a regulatory protein as well as by 4) long chain acyl-CoAs(1, 2, 3) . Kinetic evidence indicates that these two types of inhibitors bind to a site distinct from the catalytic site despite the fact that their action is competitively counteracted by glucose. The four properties mentioned above appear to be shared by all animal glucokinases(4) , suggesting that the amino acid residues participating in these regulatory functions have been conserved. Therefore, it is of interest to identify the amino acid sequences that have been specifically conserved in the glucokinase subfamily.

The sequence of glucokinase is only known for a few enzymes that are relatively close from an evolutionary point of view. These are rat liver glucokinase (5, 6) and the two human liver glucokinase isoenzymes, which show 98% identity to the rat liver enzyme(7) , and their pancreatic islet counterparts(8) . Liver and islet glucokinases are encoded by the same gene, which has two promoters and from which two types of mRNA are derived. These mRNAs differ by the first exon and give rise to two proteins that differ by 15 amino acids in their amino-terminal region(2) . Glucokinases show 32-34% identity to Saccharomyces cerevisiae hexokinases A and B (9) . The three-dimensional structure of the human beta cell glucokinase has been modeled (10) by analogy with the crystal structure of yeast hexokinase B(11) .

To determine the regions specifically conserved in the glucokinase family, we decided to clone a glucokinase that was relatively distant from mammalian glucokinase. The glucokinase from Xenopus liver seemed appropriate since the separation of amphibians from mammals probably occurred some 350 million years ago(12) .


MATERIALS AND METHODS

The source of materials and reagents was as described previously (13) . Non-recombinant Xenopus liver glucokinase and recombinant Xenopus regulatory protein were purified as described(13) . Rat liver regulatory protein and glucokinase were purified as described(14) . Peptide synthesis and purification were performed as described(15) .

Isolation and Sequencing of cDNA Clones

The Xenopus liver cDNA library constructed in ZAP I by EcoRI insertion (16) was kindly provided by Dr. Daniel R. Schoenberg (Uniformed Services, University of the Health Sciences, Bethesda, MD). Approximately 160,000 clones of this cDNA library were screened as described previously (13) using as a probe the full-length cDNA encoding rat pancreatic glucokinase(17) , which was kindly provided by Dr. M. A. Magnuson; this cDNA was excised from plasmid pGKZ9 by digestion with EcoRI, purified, and labeled with [alpha-P]dCTP by random priming(18) . The plasmid DNA of the selected clones was purified by the boiling lysis method(19) , digested with EcoRI, and analyzed by Southern blotting to identify fragments homologous to rat glucokinase. Sequencing was done on both strands after alkaline denaturation by the dideoxy method (20) using T3 and T7 primers on progressive deletion subclones generated by exonuclease III (21) or using insert-specific primers.

Construction of Expression Vector and Expression of Xenopus Liver Recombinant Glucokinase

The recombinant protein was expressed in Escherichia coli in the expression system of Studier and Moffatt(22) . PCR (^1)mutagenesis was used to introduce in pBS-XGK2.1 an NdeI restriction site at the initial ATG codon and a BamHI restriction site after the termination codon; the primers used were 5`-GTGCATATGGAAACATTTGA-3` and 5`-ACGGATCCACTAGTGACCAAT-3` (restriction sites are underlined). After purification by 1% agarose gel electrophoresis, the PCR-amplified fragment was cloned in the EcoRV site of the pBlueScript KS vector to generate pBS-XGK1374. The amplified DNA was sequenced to rule out any PCR errors. The coding region was then excised from pBS-XGK1374 with NdeI and BamHI and ligated into the expression vector pET3a to generate pET-XGK. This plasmid was used to transform the E. coli BL21(DE3) pLysS strain. These cells were grown in M9 medium containing 1% glucose, 0.1 mg/ml ampicillin, and 0.03 mg/ml chloramphenicol until the absorbance at 600 nm reached 0.6. The inducer isopropyl-1-thio-beta-D-galactopyranoside was then added to a final concentration of 0.4 mM, and the cultures were incubated in an orbital shaker for 23 h at 22 °C. Cell extracts were prepared as described previously(13, 23) . Slightly more than half of the recombinant glucokinase was soluble when the expression was performed under these conditions, whereas all of the recombinant glucokinase was present as an insoluble form when the culture and the induction were carried out at 37 °C.

Purification of Recombinant Xenopus Liver Glucokinase

For the purification of the recombinant glucokinase, a cell pellet derived from a 1-liter culture was resuspended in 50 ml of lysing buffer(13) , and the fraction of protein precipitating between 30 and 60% (NH(4))(2)SO(4) was prepared. The pellet was resuspended in 3.5 ml of buffer A (10 mM Hepes, pH 7.1, 1 mM dithiothreitol, 50 mM KCl, 10 mM glucose, and 1 µg/ml antipain) and gel-filtered on a Sephadex G-25 fine column equilibrated with buffer A. The fractions containing glucokinase activity (6 ml) were then chromatographed on a 1.6 times 13-cm DEAE-Sepharose column equilibrated with buffer A. The column was washed with 100 ml of the same buffer, and the retained glucokinase was eluted with a linear KCl gradient (20-500 mM in 2 times 100 ml of buffer A). The fractions with the highest specific activities were pooled (17 ml), and this preparation was used for kinetic investigation.

Construction of Xenopus Liver Glucokinase Mutants

The mutants of Xenopus liver glucokinase (XGK-A, XGK-B, XGK-C, XGK-D, XGK-E, and XGK-F) were constructed by an adaptation of the PCR-based site-directed mutagenesis procedure described in (24) . For each mutant, the plasmid pBS-XGK1374 was amplified using Pwo DNA polymerase and two ``back-to-back'' primers phosphorylated at their 5`-ends. One or both primers contained the mismatches needed to generate the site-directed mutations (see Table 1for primer sequences). The PCR-amplified plasmids were purified by 1% agarose gel electrophoresis, ligated, and amplified in E. coli XL1Blue. PCR errors were ruled out, and the mutations introduced were checked by sequencing the amplified DNA either 1) between the start codon (NdeI restriction site) and a unique internal NcoI restriction site (581 base pairs) for mutants XGK-A, XGK-B, XGK-C, and XGK-D or 2) between the NcoI site and the BamHI site that flanks the stop codon for mutants XGK-E and XGK-F. These nucleotide sequences were then excised from the amplified DNA and introduced in pET-XGK to replace the non-mutated sequences and to generate pET-XGK-A, pET-XGK-B, pET-XGK-C, pET-XGK-D, pET-XGK-E, and pET-XGK-F. The double mutant, pET-XGK-A-C, was constructed by inserting an NsiI-NcoI fragment of pET-XGK-A in the appropriate position in pET-XGK-C. The expression and the purification of the recombinant proteins were performed as described above.



Other Assays

Glucokinase was assayed by a pyruvate kinase/lactate dehydrogenase-coupled assay(14) . The regulatory protein was assayed by its ability to inhibit glucokinase. One unit of both rat and Xenopus regulatory protein is defined as the amount causing 50% inhibition of 15 milliunits of rat glucokinase in 1 ml in the presence of 5 mM glucose and, in the case of the rat regulator, of also 200 µM fructose 6-phosphate (25) . Protein was measured according to Bradford (26) using bovine -globulin as a standard.


RESULTS AND DISCUSSION

Isolation and Characterization of cDNAs Encoding Xenopus Liver Glucokinase

From the screening of the Xenopus liver cDNA library, three positive clones were isolated, two of which were identical. The latter, termed pBS-XGK2 and pBS-XGK7, contained an insert of 5.0 kb total length, with four EcoRI fragments of 0.24, 0.95, 0.75, and 3.0 kb. The insert of the third clone (pBS-XGK1) had a total length of 2.9 kb and contained two EcoRI fragments of 0.15 and 2.7 kb. Southern blot analysis of EcoRI-restricted plasmids revealed that the 3.0-kb fragment of pBS-XGK2 and pBS-XGK7 as well as the 2.7-kb fragment of pBS-XGK1 were homologous to the rat glucokinase cDNA.

These fragments were subcloned, and the longest, termed pBS-XGK2.1, was completely sequenced on both strands. Fig. 1shows that it has a total length of 3005 base pairs and contains an open reading frame of 1374 base pairs, encoding a protein of 458 amino acids with a predicted molecular mass of 51,154 Da (including the initiator methionine). The initiator codon (nucleotide 350) is preceded by an in-frame stop codon at nucleotide 278, indicating that the coding region of the isolated clone is complete at its 5`-end. This cDNA also contains a polyadenylation signal (nucleotides 2970-2975) followed by a short polyadenylation tail. The 2.7-kb fragment of pBS-XGK1 was partially sequenced and found to correspond to nucleotides 355-2987 of the sequence shown in Fig. 1, with a poly(A) tail of 20 nucleotides.


Figure 1: Nucleotide sequence of the cDNA encoding Xenopus liver glucokinase. The deduced amino acid sequence is indicated.



The other inserts present in clones pBS-XGK1 and pBS-XGK2 (or pBS-XGK7) presumably correspond to unrelated cDNAs that were ligated during the construction of the cDNA library. This is indicated by the fact that the glucokinase cDNAs were flanked on both sides by sequences that are typical of EcoRI linkers. Furthermore, the 0.15-kb EcoRI fragment of pBS-XGK1 contained a poly(A) tail (data not shown).

Expression of Recombinant Xenopus Glucokinase

The isolated cDNA was expressed in E. coli to verify if it encoded a protein with the properties of glucokinase. The recombinant glucokinase was purified by a procedure involving ammonium sulfate precipitation and anion-exchange chromatography. It was eluted from the DEAE-Sepharose column at a KCl concentration of 0.32 M and was nearly homogeneous at this stage. From 1 liter of culture, the yield was 800 units of glucokinase, with a specific activity of 10 units/mg of protein (25 units/mg of protein if the protein assay was carried out with bovine serum albumin as a standard); this value is comparable to those observed for other recombinant glucokinases(23, 27) .

Recombinant Xenopus glucokinase displayed a sigmoidal saturation curve for glucose, with an S(0.5) of 2.3 mM, and was inhibited competitively by palmitoyl-CoA as well as by rat and Xenopus liver regulatory proteins (see Table 1and Fig. 4). Similar results were obtained with non-recombinant Xenopus liver glucokinase (data not shown), confirming that the cloned cDNA encoded the latter enzyme.


Figure 4: Inhibition of wild-type and mutant glucokinases by Xenopus (a) and rat (b) regulatory proteins. The assays were performed with increasing concentrations of regulator in the presence of 5 mM glucose (with the exception of mutant XGK-D, which was assayed at 20 mM glucose to compensate for its lower affinity for glucose). Fructose 6-phosphate (200 µM) was added when the inhibition by the rat regulatory protein was measured.



Sequence Comparison between Glucokinases and Other Hexokinases

In Fig. 2, the sequence of Xenopus liver glucokinase has been aligned with those of rat liver and human beta cell glucokinases and with those of hexokinases that are insensitive to the regulatory protein (4) . (^2)These sequences are of the amino- and carboxyl-terminal halves of rat hexokinases I and II (28, 29, 30) and of S. cerevisiae hexokinases A and B(31) . Fig. 2also shows the secondary structure assigned to yeast hexokinase B and to human glucokinase and the amino acid residues implicated in the binding of glucose(10, 11) .


Figure 2: Alignment of the primary sequences of rat and Xenopus liver and human islet glucokinases with those of other hexokinases. Rat glucokinase (GK)(5) , the amino-terminal regions of rat hexokinases (HK) I and II (30) , the carboxyl-terminal regions of rat hexokinases I (29) and II (30) , and yeast hexokinases A and B (31) have been compared with human islet (8) and Xenopus liver glucokinases. The conserved residues are represented by dashes, and the gaps by dots; the glucose-binding amino acids are indicated by asterisks. The secondary structure elements are indicated below the sequences. Mutated residues are indicated in boldface and are underlined.



The overall identity of Xenopus glucokinase to the rat and human glucokinases is 78 and 79%, respectively. As previously observed for other glucokinases(7) , the amino-terminal end of glucokinase, corresponding to the residues encoded by the first exon, is not conserved; it is 6 and 7 amino acids shorter in the Xenopus enzyme than in its rat and human counterparts, respectively. When the sequence encoded by the remaining exons is considered, the identity to the rat and human enzymes amounts to 80 and 81%, which is much less than the 98% value observed when comparing rat to human glucokinase. As expected, the residues involved directly in glucose binding (10) are the same in Xenopus glucokinase as in other hexokinases.

To locate the conserved and nonconserved residues in the three-dimensional structure of glucokinase, a space-filling model (Fig. 3) was used because it emphasizes the surface residues, which are most likely involved in protein-protein interaction. Three different color codes identify the amino acids according to their degree of conservation. Residues that are different among glucokinases are green; these residues are most likely unimportant for glucokinase function. Those that are present in all glucokinases but not in the C-terminal half of hexokinase I or II are orange; these residues are likely to be involved in the glucokinase-specific functions, including the binding of the regulatory protein. The remaining residues are yellow; they correspond to residues that are conserved among glucokinases and in the C-terminal halves of hexokinases I and II. They are presumed to be important for hexokinase function in general, but they could also play an auxiliary role in the glucokinase-specific functions.


Figure 3: Space-filling model of human beta cell glucokinase. The space-filling rendition of the human beta cell glucokinase structure of St. Charles et al.(10) is shown. The large domain is horizontally oriented at the bottom of each panel. The residues different in the human, rat, and Xenopus glucokinases are green; residues conserved in the three glucokinases and in the C-terminal halves of rat hexokinases I and II are yellow; and the residues conserved in glucokinases but not in other mammalian hexokinases are orange. In c and d, red residues are those for which mutations resulted in a decrease in the affinity for the regulatory protein, while the blue residues represent the residues for which mutations did not cause a significant change in the affinity for the regulatory protein.



The model shows the two domains of glucokinase connected by the hinge region, which comprises the catalytic cleft (Fig. 3, a and b). It appears that the green (nonconserved) residues are spread over most of the surface of glucokinase. There is, however, a large region that is almost free of green residues: it extends on the front face of glucokinase from the tip of the small domain to the hinge region and the neighboring part of the large domain. Since this large region is made up of a number of glucokinase-specific (orange) residues, we hypothesized that it contained the binding site for the regulatory protein.

Glucokinase Mutants

To test the latter hypothesis, we have constructed several mutants (XGK-A, XGK-B, XGK-C, XGK-D, and XGK-F) of Xenopus glucokinase (see Fig. 2) in which we have replaced glucokinase-specific residues with the equivalent ones in the C-terminal half of rat hexokinase I (see Table 1). To increase the probability of having a significant effect, we chose mutations that resulted in a change in the electric charge. In addition, we have made a control mutant (XGK-E) in a region that is not conserved among glucokinases.

The mutated glucokinase proteins were all expressed and purified as described above, except for mutant XGK-F, which proved to be insoluble and could therefore not be further investigated. The kinetic properties of the five soluble mutants were compared with those of the wild-type enzyme. Table 1shows that the S(0.5) was not significantly affected for mutants XGK-A, XGK-C, and XGK-E, but that it was increased 2- and 10-fold for mutants XGK-B and XGK-D, respectively. This last mutant also showed a decrease in the sigmoidicity of the saturation curve for glucose. The decrease in affinity for glucose of mutant XGK-D is not surprising since the five consecutive mutations (Val-154 to Asp-158) introduced are close to the glucose-binding residue Ser-151 ( Fig. 2and Fig. 3)(32) . Like the wild-type enzyme, all mutants were 50% inhibited at a concentration of 2 µM palmitoyl-CoA (Table 1).

We then investigated the sensitivity of the various mutants to inhibition by the regulatory proteins both from rat liver and from Xenopus liver (Fig. 4). Mutants XGK-B, XGK-D, and XGK-E had the same sensitivity as the wild-type enzyme for rat regulatory protein and a slightly (1.5-2-fold) decreased sensitivity to Xenopus regulatory protein. In contrast, mutants XGK-A and XGK-C showed a reduction of 9- and 250-fold in their affinity for rat regulatory protein (Fig. 4b) and of 40- and 770-fold for Xenopus regulatory protein (Fig. 4a), respectively. Because of the important effect of these mutations, we also constructed the double mutant (XGK-A-C). This mutant proved to be insensitive to the highest concentration of regulatory proteins used (1 µM). Remarkably, it had conserved the same affinity for glucose and for palmitoyl-CoA as the wild-type enzyme (Table 1). Furthermore, all mutants and the wild-type protein had a similar heat stability, except for mutant XGK-E, which was half-maximally inactivated at 32 °C as compared with 35 °C (5-min incubation) for the other proteins. This result, as well as the fact that the affinities for glucose and palmitoyl-CoA and the V(max) were unchanged, indicates that the lower affinity of mutants XGK-A, XGK-C, and XGK-A-C for the regulatory protein is not due to gross structural changes(33) .

Fig. 3(c and d) shows the locations of the mutations in the glucokinase model as well as of Asn-166, a residue that, when mutated to arginine, results in a severalfold decrease in the affinity of human beta cell glucokinase for rat liver regulatory protein. (^3)The residues mutated in XGK-D, which has a lower affinity for glucose (S(0.5) = 19 mM), are in the vicinity of the catalytic cleft, whereas those mutated in XGK-F, an insoluble protein, are internal residues present inside the large domain. The residues mutated in XGK-A and XGK-C, which have a lower affinity for the regulatory protein, are situated at two extremities of the broad glucokinase-specific conserved region, whereas Asn-166 is closer to the center of this same region. The fact that region D, which lies between regions A and C, does not appear to participate in the binding of the regulatory protein suggests that the binding site for the regulatory protein is bipartite or that it has a complex shape.

A remarkable feature of the residues mutated in XGK-C, the mutant most affected in its affinity for the regulatory protein, is that they form a basic loop with two protruding side chains (His-141 and Lys-142). We have tested the possibility that a peptide of glucokinase containing residues 139-146 was able to compete with the enzyme for the inhibition by the regulatory protein. However, there was no release of inhibition at peptide concentrations up to 10M. This lack of effect could be due to the fact that the peptide does not adopt the appropriate conformation to bind to the regulatory protein (see also below).

Whether residues 51, 52, and 141-144 are directly implicated in the binding of the regulatory protein cannot be decided at present. An alternative explanation for the decreased affinity for the regulatory protein induced by the mutations could be that they result in localized changes in the three-dimensional arrangement of adjacent residues that would be directly involved in the binding of the regulatory protein. Such structural changes, if present, diminish with distance(33) , which allows us to conclude that the regulatory protein binds, if not to residues 51-52 and 141-144, to nearby residues.

The fact that the regulatory protein binds to the small domain of glucokinase and to its hinge region is in agreement with the idea that it interacts with a site distinct from the catalytic site(4) . We speculate that this binding results in competitive inhibition because it prevents the conformational changes that occur during catalysis. It is indeed well known that glucose binding induces closure of the catalytic cleft in the case of yeast hexokinase(34) . The fact that the mutations affecting the inhibition by the regulatory protein do not affect the sensitivity to palmitoyl-CoA indicates that the binding sites for these two inhibitors either are different or do not entirely coincide.


FOOTNOTES

*
This work was supported in part by the National Fund for Scientific Research, the Belgian Federal Service for Scientific, Technical, and Cultural Affairs, the Action de Recherches Concertées, the Fritz Thyssen Foundation, and the Juvenile Diabetes Foundation International. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X93494[GenBank].

§
Contributed equally to this work.

Supported by a grant from the European Economic Community in the framework of the Human Capital and Mobility Program. Chargé de Recherches of the Belgian Fonds National de la Recherche Scientifique.

**
Present address: Lab. voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.

§§
To whom correspondence should be addressed: UCL 7539, Av. Hippocrate, 75, B-1200 Brussels, Belgium. Tel.: 32-2-764-75-64; Fax: 32-2-764-75-98.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase pair(s).

(^2)
A. Vandercammen and E. Van Schaftingen, unpublished results.

(^3)
M. Veiga-da-Cunha, L. Z. Xu, Y. H. Lee, D. Marotta, S. J. Pilkis, and E. Van Schaftingen, unpublished results.


ACKNOWLEDGEMENTS

We thank Prof. H. G. Hers for helpful criticisms, K. Peel for competent technical help, and L. Bertrand for introducing us to the molecular graphics program.


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