(Received for publication, October 10, 1995; and in revised form, December 21, 1995)
From the
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.
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) .
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) .
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).
Recombinant Xenopus glucokinase displayed a sigmoidal saturation curve for glucose,
with an S 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.
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.
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 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
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. ()The residues mutated in XGK-D, which has a lower
affinity for glucose (S
= 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X93494[GenBank].