Human Glucokinase Research Articles

Structural model of human glucokinase in complex with glucose and ATP: implications for the mutants that cause hypo- and hyperglycemia.

Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Glucokinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interactions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the gamma-phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dramatic changes in the Km for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dramatically reduced the catalytic activity, as did a mutation that was predicted to disrupt an alpha-helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crystal structure for glucokinase, our models help rationalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activators and inhibitors.

Cell-biological assessment of human glucokinase mutants causing maturity-onset diabetes of the young type 2 (MODY-2) or glucokinase-linked hyperinsulinaemia (GK-HI)

Mutations in the glucokinase (GK) gene cause type-2 maturity-onset diabetes of the young type 2 (MODY-2) and GK-linked hyperinsulinaemia (GK-HI). Recombinant adenoviruses expressing the human wild-type islet GK or one of four mutant forms of GK, (the MODY-2 mutants E70K, E300K and V203A and the GK-HI mutant V455M) were transduced into glucose-responsive insulin-secreting beta-HC9 cells and tested functionally in order to initiate the first analysis in vivo of recombinant wild-type and mutant human islet GK. Kinetic analysis of wild-type human GK showed that the glucose S(0. 5) and Hill coefficient were similar to previously published data in vitro (S(0.5) is the glucose level at the half-maximal rate). E70K had half the glucose affinity of wild-type, but similar enzyme activity. V203A demonstrated decreased catalytic activity and an 8-fold increase in glucose S(0.5) when compared with wild-type human islet GK. E300K had a glucose S(0.5) similar to wild-type but a 10-fold reduction in enzyme activity. E300K mRNA levels were comparable with wild-type GK mRNA levels, but Western-blot analyses demonstrated markedly reduced levels of immunologically detectable protein, consistent with an instability mutation. V455M was just as active as wild-type GK, but with a markedly reduced S(0.5). The effects of the different GK mutants on glucose-stimulated insulin release support the kinetic and expression data. These experiments show the utility of a combined genetic, biochemical and cell-biological approach to the quantification of functional and structural changes of human GK that result from MODY-2 and GK-HI mutations.

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: enzymatic, metabolic and cationic determinants.

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.

Comparative stereochemical analysis of glucose-binding proteins for rational design of glucose-specific agents

There is a need for synthesizing glucose-sensitive molecules which can be used in glucosesensors and self-regulating insulin delivery devices. Currently, glucose-sensitive proteins, such as glucoseoxidase and concanavalin A (Con-A), are used for detecting glucose molecules. For long-term in vivoapplications, it is necessary to synthesize non-proteineous glucose-sensitive molecules which are biocompatible,nontoxic, cost-effective, and independent of environmental factors such as pH, ionic strength, orthe presence of divalent cations. As a first step toward synthesizing glucose sensitive molecules, we have compared glucose-bindingsites of four different types of glucose-binding proteins. They are human β-cell glucokinase, D-xyloseisomerase, lectins (Lathyrus ochrus isolectin I and Con-A), and glucose/ galactose binding protein. Analysisof the glucose-binding sites of their 3-dimensional crystal structures showed that the hydrogen bondsbetween the hydroxyl groups of glucose and a few types of amino acid residues of proteins provided themain attraction. In some cases, the same atom was involved in multiple hydrogen bonds. Hydrophobicinteractions between the pyranose ring of glucose and aromatic rings of hydrophobic amino acid residuesalso played an important role in the glucose specificity. A sandwich geometry was observed among thehydrophobic groups. This comparative stereochemical analysis suggests that a possible glucose bindingsite can be made by placing Asp and Asn around glucose for hydrogen bonding and Phe on both sidesof glucose for hydrophobic interaction.

Gestational diabetes mellitus and gene mutations which affect insulin secretion

We investigated whether genetic mutations known to impair insulin secretion and glucose tolerance are operative in a group of American women with gestational diabetes mellitus. Study groups were comprised of elderly non-diabetic controls ( n=55) with normal glucose tolerance and patients with gestational diabetes ( n=50), together with one family with maturity-onset diabetes of the young (three controls and three affected). No mutations were detected in any exon of the human glucokinase gene or the mitochondrial tRNA Leu(UUR) gene by single strand conformational analysis and direct exon sequencing. Also, X 2 analysis showed no significant association with gestational diabetes for a polymorphism at position −30 (G→A) of the β-cell-specific glucokinase gene promoter. We have determined that glucokinase and mitochondrial tRNA Leu(UUR) gene mutations, which are known to impair insulin secretion are relatively uncommon and do not constitute a large component of genetic risk for gestational diabetes in the study population.

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K m values around 8 mM for the substrate d-glucose with a preference for the α-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH 2-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with K i values in the range of 1 μM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC 50 value of 9 μM for alloxan and of 60 μM for ninhydrin. d-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.

Glucose-induced positive cooperativity of fructose phosphorylation by human B-cell glucokinase.

Human B-cell glucokinase displays sigmoidal kinetics towards D-glucose or D-mannose, but fails to do so towards D-fructose. Yet, D-glucose, D-mannose and 2-deoxy-D-glucose confer to the enzyme positive cooperativity towards D-fructose. For instance, in the presence of 5 mM D-[U-14C]fructose, its rate of phosphorylation is increased to 214.3 +/- 11.0%, 134.0 +/- 4.3% and 116.5 +/- 3.0% of paired control value by D-glucose, D-mannose and 2-deoxy-D-glucose (each 6 mM), respectively. D-glucose and, to a lesser extent, D-mannose also display reciprocal kinetic cooperativity. D-fructose, however, fails to affect D-glucose or D-mannose phosphorylation under conditions in which positive cooperativity is otherwise observed. These findings are relevant to the reciprocal effects of distinct hexoses upon their phosphorylation by B-cell glucokinase and, as such, to the metabolic and functional response evoked in pancreatic islet B-cells by these sugars, when tested either separately or in combination.

The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells

The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells

Liver-specific Enhancer of the Glucokinase Gene

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.

The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells.

The glycolytic enzyme glucokinase plays a primary role in the glucose-responsive secretion of insulin, and defects of this enzyme can cause NIDDM. As a step toward understanding the molecular basis of glucokinase (GK) gene regulation, we assessed the structure and regulation of the human GK gene beta-cell-type promoter. The results of reporter gene analyses using HIT-T15 cells revealed that the gene promoter was comprised of multiple cis-acting elements, including two primarily important cis-motifs: a palindrome structure, hPal-1, and the insulin gene cis-motif A element-like hUPE3. While both elements were bound specifically by nuclear proteins, it was the homeodomain-containing transcription factor insulin promoter factor 1 (IPF1)/STF-1/PDX-1 that bound to the hUPE3 site: IPF1, when expressed in CHO-K1 cells, became bound to the hUPE3 site and activated transcription. An anti-IPF1 antiserum used in gel-mobility shift analysis supershifted the DNA protein complex formed with the hUPE3 probe and nuclear extracts from HIT-T15 cells, thus supporting the involvement of IPF1 in GK gene activation in HIT-T15 cells. In contrast to the insulin gene, however, neither the synergistic effect of the Pan1 expression on the IPF1-induced promoter activation nor the glucose responsiveness of the activity was observed for the GK gene promoter. These results revealed some conservative but unique features for the transcriptional regulation of the beta-cell-specific genes in humans. Being implicated in insulin and GK gene regulations as a common transcription factor, IPF1/STF-1/PDX-1 is likely to play an essential role in maintaining normal beta-cell functions.

Promoter Variation in the Liver Glucokinase Is a Risk Factor for Non-Insulin-Dependent Diabetes Mellitus

We have previously reported a common variation in the liver promoter of the human glucokinase, which is regulated by insulin, in the patients with non-insulin-dependent diabetes mellitus (NIDDM). The variation occurred within a 10-bp region completely conserved between human and rat. Its basic motif was almost identical to the insulin regulatory element of the phosphoenolpyruvate carboxykinase gene.In vitrotransfection experiment showed that the G-to-A variation causes a 58% reduction in the promoter activity. After oral glucose challenge, the homozygous A/A subjects had the highest stimulated insulin levels at 60 and 90 minutes and the highest insulin area under the curve as compared to the subjects with other genotypes, which suggested the homozygous A/A subjects were more insulin resistant. As insulin resistance is a risk factor of NIDDM, we concluded that this promoter variation is a risk factor for NIDDM.

Cloning and Characterization of the Mouse Glucokinase Gene Locus and Identification of Distal Liver-Specific DNase I Hypersensitive Sites

We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1β and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from ∼−9.2 to ∼+15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from −335 to +736 bp (1071 bp), relative to the transcription start site in β cells. These sequences revealed that mouse GK is >94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-spe- cific expression of GK to be performed.

Human glucokinase regulatory protein (GCKR): cDNA and genomic cloning, complete primary structure, and chromosomal localization.

Null mutations in the glucokinase (GCK) gene can cause autosomal dominant type 2 diabetes (maturity onset diabetes of the young, MODY); however, MODY is genetically heterogeneous. In both liver and pancreatic islet, glucokinase is subject to inhibition by a regulatory protein (GCKR). Given the role of GCK in MODY, GCKR is itself a candidate type 2 diabetes susceptibility gene. Here we describe the structure of full-length (2.2 kb) cDNA for human GCKR, from the hepatoblastoma cell line HepG2. The human GCKR translation product has 625 amino acids and a predicted molecular weight of 68,700. It has 88% amino acid identity to rat GCKR. Yeast artificial chromosomes (YAC clones) containing human GCKR were isolated, and the gene was mapped to Chromosome (Chr) 2p23 by fluorescent in situ hybridization and somatic cell hybrid analysis.

Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme.

Mutations in the human glucokinase (GK) gene are thought to cause maturity-onset diabetes of youth (MODY) by leading to the production of enzymes with reduced catalytic activities and increased glucose Km values. However, in some cases the diabetic phenotype is more severe than might be predicted from these apparent kinetic effects alone. To determine whether these mutations might also effect other characteristics of the enzyme, nine MODY-associated mutants were expressed as fusion proteins with Schistosoma japonicum glutathione S-transferase (GST) and compared with three wild-type human GK isoforms that were also expressed in the same manner. Three GST-GK isoforms (liver 1, liver 2 and islet) were kinetically indistinguishable from each other and from purified rat liver GK. Noteworthy is a glucose-induced fit effect for the interaction of trinitrophenyl (TNP)-ATP with GST-GK, whereby glucose significantly increased the affinity of TNP-ATP binding to GST-GK without changing the stoichiometry of binding. The nine MODY-associated mutations studied either showed diminished catalytic activity, substrate affinities, allosteric regulation, or stability of the fusion enzyme. We conclude that: (1) Gly261 and Lys414 are important for ATP binding; (2) Val203 may be essential for a glucose-induced fit effect; and (3) the stability of fusion protein may be significantly reduced when Glu300 is replaced by Lys. These results suggest that, in addition to effects on the Km and Vmax. of GK, a decrease in the ATP-binding affinity or stability of the mutated enzyme may also contribute to a reduction of GK activity in individuals with GK-MODY. In the B-cell this would have the effect of blunting glucose-stimulated insulin release, thereby contributing to the diabetic phenotype.

Human β-Cell Glucokinase: DUAL ROLE OF SER-151 IN CATALYSIS AND HEXOSE AFFINITY

Glucokinase is distinguished from yeast hexokinase and low Km mammalian 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 Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values 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 Km for 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.

Author Index for Volume 23

Previous reports concerning the regulation of glucokinase expression in β cells have been done using cell models from rodent origin. Evidence is lacking so far to implicate the same regulatory mechanisms in human cells. In this study, we investigate the effects of glucose on the expression of glucokinase using isolated human pancreatic islets. High glucose (16.7 mM), in a time-dependent manner, increases the amount of immunoreactive glucokinase (+150% after 7 days culture, P < 0.01) without apparent changes in glucokinase gene expression, suggesting that glucose exerts its effect at a posttranscriptional level. Mannose, but not the nonmetabolized hexoses, 3-O-methylglucose or 2-deoxyglucose, increases glucokinase protein content. Even though these findings are compatible with an involvement of signals derived from glucose metabolism, additional data argue against this hypothesis: (i) a glucokinase inhibitor (mannoheptulose) does not block glucose-induced increase in glucokinase content and (ii) other metabolic fuels (amino acids) are ineffective. We suggest that the glucose molecule, by mechanisms yet to be defined, but probably not involving its metabolism, regulates human glucokinase expression.

Polymorphic Microsatellite Repeat Markers at the Glucokinase Gene Locus Are Positively Associated With NIDDM in Japanese

To assess the possible role of glucokinase defects contributing to a genetic susceptibility to NIDDM in Japanese, allelic frequencies of two microsatellite repeat polymorphisms, one in the 3'-flanking region (GCK1) and the other in the 5'-flanking region (GCK2) of the human glucokinase gene, were analyzed in subjects with NIDDM and in nondiabetic control subjects. After typing 107 diabetic and 74 nondiabetic subjects, we found four GCK1 alleles (Z, Z2, Z4, Z6) and six GCK2 alleles (0, -4, -2, 2, 4, 8). The frequency distribution of GCK1 alleles was different between the two groups (P = 0.005), although not significant after correction for multiple comparisons. The Z4 allele was found more frequently in diabetic than in nondiabetic subjects (23 vs. 10%, P = 0.002). This was still significant after correction for multiple comparisons (P < 0.05). The frequency distribution of GCK2 alleles was not different between the two groups. However, the -2 allele was more common in diabetic than in nondiabetic subjects (P = 0.044), although not significant after adjusting for multiple comparisons. Clinical characteristics were compared between the diabetic subjects with Z4 and/or -2 allele and those without either of these two alleles. No differences were found in the age of diagnosis, positive family history, mode of therapy, current HbA1c, or daily urinary C-peptide immunoreactivity excretion between the two groups. We demonstrated a significant association between GCK1 and GCK2 alleles and NIDDM. The results indicate that the polymorphic alleles GCK1 and GCK2 could be genetic markers in NIDDM in Japanese, suggesting a relationship between glucokinase defects and the susceptibility to NIDDM in this population.

Two microsatellite repeat polymorphisms flanking opposite ends of the human glucokinase gene: use in haplotype analysis of Welsh Caucasians with type 2 (non-insulin-dependent) diabetes mellitus.

The purpose of this study was to evaluate the role of potential glucokinase defects contributing to susceptibility to Type 2 (non-insulin-dependent) diabetes mellitus in Welsh Caucasians. For this analysis, two microsatellite repeat polymorphisms flanking opposite ends of the gene were employed. For a recently described microsatellite (GCK2), located 6 kilobases upstream of islet exon 1, six different sized alleles were observed, with heterozygosity of 0.50 and polymorphism information content 0.44. Combined heterozygosity with another microsatellite repeat (GCK1) was 0.72. Significant linkage disequilibrium was noted between GCK2 and GCK1, suggesting that haplotypes may be a better predictor of Type 2 diabetes than analysis with either microsatellite alone. Using these two markers, the association with Type 2 diabetes was examined. The frequencies of alleles and genotypes at GCK1 did not differ between the patients with Type 2 diabetes (n = 157) and control subjects (n = 73). Similarly no differences were observed in GCK2 alleles or genotypes. The frequencies of haplotypes, derived from the two markers, also did not differ between the two groups. To investigate the possibility of minor metabolic effects of glucokinase defects, we also studied the association between the GCK alleles or haplotypes and the response profiles to meal tolerance tests. No association was observed between plasma glucose or insulin responses to meal tolerance tests with GCK haplotypes or alleles. These results suggest that glucokinase mutations in Welsh Caucasians are not major determinants of susceptibility to the common type of Type 2 diabetes.

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.

Characterization of a third simple tandem repeat polymorphism in the human glucokinase gene.

Recent studies have implicated mutations in the glucokinase gene as a cause of early-onset Type 2 (non-insulin-dependent) diabetes mellitus. Two simple tandem repeat DNA polymorphisms have been identified in this gene and used for genetic studies. However, their heterozygosity is relatively low and not all families are informative for linkage with these markers. Here we report the characterization of a third simple tandem repeat polymorphism that can be used for genetic studies.