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Insights into the Structure and Regulation of Glucokinase from a Novel Mutation (V62M), Which Causes Maturity-onset Diabetes of the Young

2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês

10.1074/jbc.m413146200

ISSN

1083-351X

Autores

Anna L. Gloyn, Stella Odili, Dorothy Zelent, Carol Buettger, Harriet Castleden, Anna Steele, Amanda Stride, Chyio Shiota, Mark A. Magnuson, Renata Lorini, Giuseppe d’Annunzio, Charles A. Stanley, Jae Kwagh, Emile Van Schaftingen, Maria Veiga‐da‐Cunha, Fabrizio Barbetti, Pete Dunten, Yi Han, Joseph Grimsby, Rebecca Taub, Sian Ellard, Andrew T. Hattersley, Franz M. Matschinsky,

Tópico(s)

Diabetes and associated disorders

Resumo

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mm). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of β-cells and hepatocytes. Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mm). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of β-cells and hepatocytes. Glucokinase (GCK) 1The abbreviations used are: GCK, glucokinase; GKRP, glucokinase regulatory protein; MODY, maturity-onset diabetes of the young; fpg, fasting plasma glucose; GST, glutathionyl S-transferase; S6P, sorbitol 6-phosphate; WT, wild type. plays a critical role in the regulation of insulin secretion and has been termed the pancreatic β-cell glucose sensor on account of its kinetics, which allow the β-cells to change glucose phosphorylation rate over a range of physiological glucose concentrations. These kinetic characteristics are the enzyme's low affinity for glucose (S0.5 ∼ 7.5 mm), cooperativity with glucose (Hill number of ∼1.7), and lack of inhibition by its product glucose 6-phosphate. Glucokinase plays an important role in glucose sensing not only in the pancreatic β-cell but also in the liver and a variety of neural/neuroendocrine cells. These include the pancreatic α-cell, L- and K-type gut enterocytes, and certain rare neurons in the central nervous system, mainly in the hypothalamus (1.Cheung A.T. Dayanandan B. Lewis J.T. Korbutt G.S. Rajotte R.V. Bryer-Ash M. Boylan M.O. Wolfe M.M. Kieffer T.J. Science. 2000; 290: 1959-1962Crossref PubMed Scopus (259) Google Scholar, 2.Jetton T.L. Liang Y. Pettepher C.C. Zimmerman E.C. Cox F.G. Horvath K. Matschinsky F.M. Magnuson M.A. J. Biol. Chem. 1994; 269: 3641-3654Abstract Full Text PDF PubMed Google Scholar, 3.Schuit F.C. Huypens P. Heimberg H. Pipeleers D.G. Diabetes. 2001; 50: 1-11Crossref PubMed Scopus (316) Google Scholar). It is the sum of its actions in these multiple sites that ultimately determines the blood glucose concentration. In the liver glucokinase is regulated by glucokinase regulatory protein (GKRP), which acts as a competitive inhibitor with respect to glucose (4.Van Schaftingen E. Eur. J. Biochem. 1989; 179: 179-184Crossref PubMed Scopus (161) Google Scholar, 5.Van Schaftingen E. Detheux M. Veiga da Cunha M. FASEB J. 1994; 8: 414-419Crossref PubMed Scopus (206) Google Scholar). In addition to this role GKRP also determines the subcellular location of glucokinase within the liver cell (6.Toyoda Y. Tsuchida A. Iwami E. Shironoguchi H. Miwa I. Horm. Metab. Res. 2001; 33: 329-336Crossref PubMed Scopus (21) Google Scholar). Glucokinase translocates between the nucleus and the cytoplasm depending on the metabolic state of the cells. When glucokinase is not bound to hepatic GKRP and therefore present in the cytoplasm, the enzyme facilitates hepatic glucose utilization and glycogen synthesis thereby helping to lower the blood glucose concentration. Consistent with this role in the liver, mice that totally lack hepatic glucokinase have impaired glucose tolerance (7.Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 305-315Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar). There is controversy in the literature as to whether GKRP is expressed in the β-cell (8.Grimsby J. Coffey J.W. Dvorozniak M.T. Magram J. Li G. Matschinsky F.M. Shiota C. Kaur S. Magnuson M.A. Grippo J.F. J. Biol. Chem. 2000; 275: 7826-7831Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9.Zawalich W.S. Rognstad R. Pagliara A.S. Matschinsky F.M. J. Biol. Chem. 1977; 252: 8519-8523Abstract Full Text PDF PubMed Google Scholar, 10.Alvarez E. Roncero I. Chowen J.A. Vazquez P. Blazquez E. J. Neurochem. 2002; 80: 45-53Crossref PubMed Scopus (56) Google Scholar) and whether glucokinase translocates from the nucleus to the cytoplasm during glucose stimulation (11.Rizzo M.A. Magnuson M.A. Drain P.F. Piston D.W. J. Biol. Chem. 2002; 277: 34168-34175Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12.Arden C. Harbottle A. Baltrusch S. Tiedge M. Agius L. Diabetes. 2004; 53: 2346-2352Crossref PubMed Scopus (41) Google Scholar). Inactivating mutations in the gene encoding this enzyme (GCK) cause a subtype of maturity-onset diabetes of the young (MODY2/GCK-MODY) (13.Hattersley A.T. Turner R.C. Permutt M.A. Patel P. Tanizawa Y. Chiu K.C. O'Rahilly S. Watkins P.J. Wainscoat J.S. Lancet. 1992; 339: 1307-1310Abstract PubMed Scopus (325) Google Scholar, 14.Froguel P. Vaxillaire M. Sun F. Velho G. Zouali H. Butel M.O. Lesage S. Vionnet N. Clement K. Fougerousse F. Tanizawa Y. Weissenbach J. Beckmann J.S. Lathrop G.M. Passa P. Permutt M.A. Cohen D. Nature. 1992; 356: 162-164Crossref PubMed Scopus (567) Google Scholar). To date over 190 mutations have been reported in the literature (15.Gloyn A.L. Hum. Mutat. 2003; 22: 353-362Crossref PubMed Scopus (231) Google Scholar). These mutations include non-sense, frameshift, deletions, splice-site, and missense mutations, and they are distributed throughout the ten exons (1a-10) of the pancreatic isoform of the gene. The functional characterization of over 35 missense GCK mutations that cause MODY, has shown that a number of these kinetic parameters are altered and that usually more than one parameter is changed (16.Gloyn A. Odili S. Buettger C. Njolstad P.R. Shiota C. Magnuson M. Matschinsky F. Matschinsky F.M. Magnuson M.A. Glucokinase and Glycemic Diseases: From the Basics to Novel Therapeutics. 16. Karger, Basel, Switzerland2004: 92-109Google Scholar, 17.Davis E.A. Cuesta-Munoz A. Raoul M. Buettger C. Sweet I. Moates M. Magnuson M.A. Matschinsky F.M. Diabetologia. 1999; 42: 1175-1186Crossref PubMed Scopus (120) Google Scholar, 18.Miller S.P. Anand G.R. Karschnia E.J. Bell G.I. LaPorte D.C. Lange A.J. Diabetes. 1999; 48: 1645-1651Crossref PubMed Scopus (70) Google Scholar). These changes usually include an increase in the glucose S0.5 and/or a decrease in the turnover of the enzyme (kcat). This exploration has also uncovered mutants that are thermally labile (19.Matschinsky F.M. Diabetes. 2002; 51: S394-S404Crossref PubMed Google Scholar). These mutations may have additional defects or appear kinetically close to normal or entirely normal (19.Matschinsky F.M. Diabetes. 2002; 51: S394-S404Crossref PubMed Google Scholar). The stability of the mutant glutathionyl S-transferase (GST)-E300K-GCK has been studied extensively in a cell biological assessment of enzyme expression and stability in addition to in vitro thermolability assays providing evidence that instability is indeed pathogenic in this case (20.Burke C.V. Buettger C.W. Davis E.A. McClane S.J. Matschinsky F.M. Raper S.E. Biochem. J. 1999; 342: 345-352Crossref PubMed Google Scholar). Mutations in GCK not only cause hyperglycemia (MODY), they can also result in hypoglycemia, hyperinsulinemia of infancy (21.Gloyn A.L. Noordam K. Willemsen M.A. Ellard S. Lam W.W. Campbell I.W. Midgley P. Shiota C. Buettger C. Magnuson M.A. Matschinsky F.M. Hattersley A.T. Diabetes. 2003; 52: 2433-2440Crossref PubMed Scopus (139) Google Scholar, 22.Glaser B. Kesavan P. Haymen M. Davies E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (519) Google Scholar, 23.Cuesta-Munoz A.L. Huopio H. Otonkoski T. Gomez-Zumaquero J.M. Nanto-Salonen K. Rahier J. Lopez-Enriquez S. Garcia-Gimeno M.A. Sanz P. Soriguer F.C. Laakso M. Diabetes. 2004; 53: 2164-2168Crossref PubMed Scopus (165) Google Scholar, 24.Christesen H.B. Jacobsen B.B. Odili S. Buettger C. Cuesta-Munoz A. Hansen T. Brusgaard K. Massa O. Magnuson M.A. Shiota C. Matschinsky F.M. Barbetti F. Diabetes. 2002; 51: 1240-1246Crossref PubMed Scopus (138) Google Scholar). Five missense mutations have been reported (T65I, W99R, Y214C, V455M, and A456V), and functional characterization of these mutations has shown that they are activating mutations, because there is a decrease in the glucose S0.5 and/or an increase in the kcat (21.Gloyn A.L. Noordam K. Willemsen M.A. Ellard S. Lam W.W. Campbell I.W. Midgley P. Shiota C. Buettger C. Magnuson M.A. Matschinsky F.M. Hattersley A.T. Diabetes. 2003; 52: 2433-2440Crossref PubMed Scopus (139) Google Scholar, 22.Glaser B. Kesavan P. Haymen M. Davies E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (519) Google Scholar, 23.Cuesta-Munoz A.L. Huopio H. Otonkoski T. Gomez-Zumaquero J.M. Nanto-Salonen K. Rahier J. Lopez-Enriquez S. Garcia-Gimeno M.A. Sanz P. Soriguer F.C. Laakso M. Diabetes. 2004; 53: 2164-2168Crossref PubMed Scopus (165) Google Scholar, 24.Christesen H.B. Jacobsen B.B. Odili S. Buettger C. Cuesta-Munoz A. Hansen T. Brusgaard K. Massa O. Magnuson M.A. Shiota C. Matschinsky F.M. Barbetti F. Diabetes. 2002; 51: 1240-1246Crossref PubMed Scopus (138) Google Scholar). In a structural model of GCK (25.Mahalingam B. Cuesta-Munoz A. Davis E.A. Matschinsky F.M. Harrison R.W. Weber I.T. Diabetes. 1999; 48: 1698-1705Crossref PubMed Scopus (60) Google Scholar) these mutations have been shown to cluster in a region remote from the substrate binding site termed the allosteric activator site (21.Gloyn A.L. Noordam K. Willemsen M.A. Ellard S. Lam W.W. Campbell I.W. Midgley P. Shiota C. Buettger C. Magnuson M.A. Matschinsky F.M. Hattersley A.T. Diabetes. 2003; 52: 2433-2440Crossref PubMed Scopus (139) Google Scholar). Recently, a novel class of small molecular activators of GCK has been described (26.Grimsby J. Sarabu R. Corbett W. Haynes N. Bizzarro F. Coffey J. Guertin K. Hillard D. Kester R. Mahaney P. Marcus L. Qi L. Spence C. Tengi J. Magnuson M. Matschinsky F. Grippo J.F. Science. 2003; 301: 370-373Crossref PubMed Scopus (439) Google Scholar, 27.Kamata K. Mitsuya M. Nishimura T. Eiki J. Nagata Y. Structure (Camb.). 2004; 12: 429-438Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 28.Brocklehurst K.J. Payne V.A. Davies R.A. Carroll D. Vertigan H.L. Wightman H.J. Aiston S. Waddell I.D. Leighton B. Coghlan M.P. Agius L. Diabetes. 2004; 53: 535-541Crossref PubMed Scopus (133) Google Scholar); these have been shown to lower blood glucose levels in rodent models of type 2 diabetes (26.Grimsby J. Sarabu R. Corbett W. Haynes N. Bizzarro F. Coffey J. Guertin K. Hillard D. Kester R. Mahaney P. Marcus L. Qi L. Spence C. Tengi J. Magnuson M. Matschinsky F. Grippo J.F. Science. 2003; 301: 370-373Crossref PubMed Scopus (439) Google Scholar). These molecules act at the allosteric activator site and have a similar effect to the naturally occurring activating GCK mutations (26.Grimsby J. Sarabu R. Corbett W. Haynes N. Bizzarro F. Coffey J. Guertin K. Hillard D. Kester R. Mahaney P. Marcus L. Qi L. Spence C. Tengi J. Magnuson M. Matschinsky F. Grippo J.F. Science. 2003; 301: 370-373Crossref PubMed Scopus (439) Google Scholar, 27.Kamata K. Mitsuya M. Nishimura T. Eiki J. Nagata Y. Structure (Camb.). 2004; 12: 429-438Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Given the existence of this allosteric activator site and the fact that GCK responds to this novel class of pharmacological activators, an endogenous allosteric activator of GCK has been proposed (19.Matschinsky F.M. Diabetes. 2002; 51: S394-S404Crossref PubMed Google Scholar, 21.Gloyn A.L. Noordam K. Willemsen M.A. Ellard S. Lam W.W. Campbell I.W. Midgley P. Shiota C. Buettger C. Magnuson M.A. Matschinsky F.M. Hattersley A.T. Diabetes. 2003; 52: 2433-2440Crossref PubMed Scopus (139) Google Scholar). Recently the crystal structure of glucokinase has been solved in its free and liganded forms (22.Glaser B. Kesavan P. Haymen M. Davies E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (519) Google Scholar, 23.Cuesta-Munoz A.L. Huopio H. Otonkoski T. Gomez-Zumaquero J.M. Nanto-Salonen K. Rahier J. Lopez-Enriquez S. Garcia-Gimeno M.A. Sanz P. Soriguer F.C. Laakso M. Diabetes. 2004; 53: 2164-2168Crossref PubMed Scopus (165) Google Scholar), which has confirmed the presence of an allosteric activator site and demonstrated that there are global conformation changes, which could be the structural corollary of the mnemonical mechanism that is invoked to explain the cooperative kinetics of the enzyme with glucose (27.Kamata K. Mitsuya M. Nishimura T. Eiki J. Nagata Y. Structure (Camb.). 2004; 12: 429-438Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). In this study we present a novel GCK mutation (V62M), which has been identified in two families and shown to co-segregate with MODY. When functionally characterized, recombinant GST-V62M GCK is paradoxically mildly activating rather than inactivating. This study provides the most detailed functional assessment of a GCK mutation to date and includes extensive mutagenesis studies at residue Val62, which increase our structural insights and enhance our understanding of the allosteric activator site. Family 1 (Exeter, UK)—The female proband (III:4) presented with gestational diabetes during her first pregnancy. This diagnosis was made on the basis of an oral glucose tolerance test performed at 26-week gestation, which showed a fasting value of 5.5 and a 2-h value of 9.2 mm. She was managed on diet alone. Her male infant was born at 41 weeks (birth weight, 3.27 kg; length, 50 cm; head circumference, 35 cm; percentiles were 16th, 17th, and 27th, respectively). An oral glucose tolerance test performed on the proband at 8 weeks post partum showed a fasting level of 6.5 and a 2-h value of 7.1 mm. The proband's father (II:6) was diagnosed with abnormal glucose tolerance at the age of 57 years during routine screening and had been treated by diet. The deceased paternal grandmother had diabetes. Anthropometrical measurements (height, weight, body mass index), fasting blood glucose (fpg), lipids, and HbA1c were measured in all family members available for testing; including one brother, two paternal aunts, one paternal uncle, and four paternal first cousins (see Fig. 1a). Family 2 (Italy)—The male proband (IV:1) and his identical twin brother (IV:2) were diagnosed with MODY on the basis of elevated fpg values at the age of 6 years. Both boys were born at 39 weeks (birth weight, 3.3 kg). Details of this family are shown in Fig. 1b. Genomic DNA was extracted from peripheral lymphocytes using a Wizard DNA extraction kit (Promega, Southampton, UK). The coding regions of exons 1a-10 and the intron-exon boundaries of the glucokinase (GCK) gene were amplified by PCR using published primer sequences (29.Stoffel M. Froguel P. Takeda J. Zouali H. Vionnet N. Nishi S. Weber I.T. Harrison R.W. Pilkis S.J. Lesage S. Vaxillaire M. Velho G. Sun F. Iris F. Passa P. Cohen D. Bell G.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7698-7702Crossref PubMed Scopus (223) Google Scholar). PCR products were purified using QIAquick PCR purification columns (Qiagen), and both strands were sequenced using a BigDye terminator cycle sequencing kit (Applied Biosystems, Warrington, UK) according to the manufacturer's recommendations. Reactions were analyzed on an ABI 3100 DNA sequencer (Applied Biosystems). Recombinant human islet wild type enzyme and the mutants V62A, V62E, V62F, V62K, V62L, V62M, V62Q, V62T, and E300K were generated using methods previously described (30.Liang Y. Kesavan P. Wang L.Q. Niswender K. Tanizawa Y. Permutt M.A. Magnuson M.A. Matschinsky F.M. Biochem. J. 1995; 309: 167-173Crossref PubMed Scopus (74) Google Scholar). The enzymes were expressed in the form of GST fusion proteins using the protocols developed during the study of GST-GCK V455M and other GCK mutations (17.Davis E.A. Cuesta-Munoz A. Raoul M. Buettger C. Sweet I. Moates M. Magnuson M.A. Matschinsky F.M. Diabetologia. 1999; 42: 1175-1186Crossref PubMed Scopus (120) Google Scholar). The following modifications to the protocol were made. Protocol A was carried out with 11 glucose dilutions between 0 and 100 mm for each mutant and WT GST-GCK. Protocol B was carried out with glucose at ×10 S0.5 for wild type, each Val62 mutant and E300K GCK. Further kinetic analysis was performed in the presence of the newly discovered compounds (RO028165, RO0274375, and RO0283946; Fig. 2) that allosterically activate wild type GCK (26.Grimsby J. Sarabu R. Corbett W. Haynes N. Bizzarro F. Coffey J. Guertin K. Hillard D. Kester R. Mahaney P. Marcus L. Qi L. Spence C. Tengi J. Magnuson M. Matschinsky F. Grippo J.F. Science. 2003; 301: 370-373Crossref PubMed Scopus (439) Google Scholar). Experiments were performed in the presence of 0.3 μm, 1 μm, 3 μm, 9 μm, 27 μm, and 60 μm RO0281675, RO0274375, and RO0283946. The results were compared with those obtained with wild type GST-GCK preparations freshly made for the present investigation. The activity index, an expression of the proposed in situ phosphorylation capacity of the enzyme, was calculated as previously described (24.Christesen H.B. Jacobsen B.B. Odili S. Buettger C. Cuesta-Munoz A. Hansen T. Brusgaard K. Massa O. Magnuson M.A. Shiota C. Matschinsky F.M. Barbetti F. Diabetes. 2002; 51: 1240-1246Crossref PubMed Scopus (138) Google Scholar). Kinetic analysis was also performed with human and rat recombinant glucokinase regulatory protein (GKRP). GKRP is a competitive inhibitor of glucose (31.Veiga-da-Cunha M. Xu L.Z. Lee Y.H. Marotta D. Pilkis S.J. Van Schaftingen E. Diabetologia. 1996; 39: 1173-1179Crossref PubMed Scopus (34) Google Scholar), and consequently the kinetic analysis was carried out as previously described with glucose at 3 mm for wild type, V62F, and V62Q and to account for the decreased or increased glucose S0.5 values at 1 mm for V62L, 1.5 mm for V62M, 2.5 mm for V62K, 4.5 mm for V62E, 7.5 mm for V62T, and 8.5 mm for V62A (17.Davis E.A. Cuesta-Munoz A. Raoul M. Buettger C. Sweet I. Moates M. Magnuson M.A. Matschinsky F.M. Diabetologia. 1999; 42: 1175-1186Crossref PubMed Scopus (120) Google Scholar). Thermal stability of the mutant V62M-GST GCK enzyme, the known instability mutant E300K-GST GCK, and wild type GST-GCK was tested using protocols previously described (32.Kesavan P. Wang L. Davis E. Cuesta A. Sweet I. Niswender K. Magnuson M.A. Matschinsky F.M. Biochem. J. 1997; 322: 57-63Crossref PubMed Scopus (44) Google Scholar). The following modifications were made: enzyme stock solutions were diluted in storage buffer containing either 0 or 50 mm glucose. The enzymes were incubated in a water bath at 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, and 52.5 °C for 30 min. Glucokinase activity was then determined spectrophotometrically as described above. Glucose has been shown to stabilize glucokinase (33.Meglasson M.D. Matschinsky F.M. Diabetes/Metabolism Reviews. 1986; 2: 163-214Crossref PubMed Scopus (416) Google Scholar). The stabilization of mutant V62M, E300K, and wild type GCK by the addition of glucose was therefore investigated. Briefly, enzyme stock solutions lacking glucose were diluted in buffer with increasing concentrations of glucose ranging from 0 to ∼100 mm and to achieve comparable protein concentrations of ∼75 μg/ml. The enzymes were incubated in a water bath at 42.5 °C for 30 min. Glucokinase activity was then determined spectrophotometrically as described above. Structural analysis of V62M and published activating mutations in the glucokinase enzyme was performed using a homology model based on the WT-GCK crystal structure, co-cystallized with α-d-glucose and the allosteric activator (RO0283946), as a template (42.Corbett, W. L., Crowther, R. L., Dunten, P. W., Kammlott, R. U., and Lukacs, C. M. (August 20, 2003) Crystal Structure Patent GB2385328Google Scholar). In addition, homology models for each of the mutations at amino acid position 62 were built. Interaction energies between the amino acids comprising the activator binding site and each of the pharmacological activators (RO028165, RO0274375, and RO0283946) in its x-ray position were calculated using MOE 2Chemical Computing Group Inc., Quebec, Canada. software and MMFF94X force fields. Sequencing of Glucokinase Gene—Direct sequencing of the entire coding region and exon-intron boundaries of the glucokinase gene was performed in the probands from both families. In both families a heterozygous missense mutation substituting methionine for valine at codon 62 (V62M; GTG>ATG) in exon 2 was identified. In family 1 (UK), this mutation was also identified in the proband's father, brother, paternal aunt, and cousin (Fig. 1a) and co-segregated with hyperglycemia as shown by either a raised fasting glucose of >5.5 mm or an elevated HbA1c above the normal range. In family 2 (Italy), the mutation was identified in the proband, the proband's identical twin brother, and the proband's mother. The other affected family members were not available for testing (Fig. 1b). This mutation was not found in over 100 normal chromosomes. Biochemical Characterization of V62M—The mutant enzyme was expressed as a GST fusion protein, and the purified enzyme was subjected to kinetic analysis (kcat, glucose S0.5, nH, and ATP Km) (Table I). Four preparations of wild type, mutant V62M, and four of E300K GST-GCK were purified. All GCK proteins analyzed were found to be essentially pure as indicated by the presence of a single band at 75 kDa on phast gel (Amersham Biosciences) electrophoresis (data not shown). The functional data are shown in Table I (except those for E300K, which have been published already (12.Arden C. Harbottle A. Baltrusch S. Tiedge M. Agius L. Diabetes. 2004; 53: 2346-2352Crossref PubMed Scopus (41) Google Scholar, 13.Hattersley A.T. Turner R.C. Permutt M.A. Patel P. Tanizawa Y. Chiu K.C. O'Rahilly S. Watkins P.J. Wainscoat J.S. Lancet. 1992; 339: 1307-1310Abstract PubMed Scopus (325) Google Scholar)). The mutant enzyme showed a significant increase in affinity for glucose indicated by the decrease in glucose S0.5 value (4.88 ± 0.25 versus 7.55 ± 0.23 V62M and WT, respectively). Paradoxically, the change to the kinetic parameters of this mutant result in an increase in the activity of this enzyme compared with wild type of ∼4-fold inconsistent with this mutation causing MODY.Table IMutational Analysis of the V62 GKA binding site Data are means ± S.E. for wild type and V62M GCK. The results are the means of the kinetic analysis of four independent expressions of wild type and mutant V62M GST-GCK. For the remaining mutants two independent expressions were prepared and the mean and individual data points are shown.MutantsParametersYieldProtein concentrationkcatGlucose S0.5ATP KmnHGIaGI, the activity index for the enzyme was calculated as previously described (24)EC50 for RO0274377mg/litermg/mls−1mmμmWT41.3 ± 3.333.03 ± 0.1662.3 ± 4.757.55 ± 0.230.41 ± 0.331.74 ± 0.041.45 ± 0.116.85 ± 0.43V62A23.1 (20.6, 25.6)1.56 (1.37, 1.75)43.1 (48.8, 37.3)27.4 (24.6, 30.2)0.20 (0.20, 0.20)1.51 (1.50, 1.51)0.34 (0.38, 0.29)13.2 (10.0, 16.3)V62E5.64 (4.61, 6.66)0.50 (0.48, 0.52)4.92 (5.22, 4.62)19.5 (23.8, 15.1)0.07 (0.08, 0.05)1.15 (1.31, 0.98)0.16 (0.16, 0.15)NAbNA, not applicableV62F17.4 (21.2, 13.5)1.08 (1.22, 0.94)33.7 (39.4, 28.0)8.93 (9.48, 8.37)0.71 (0.70, 0.71)1.43 (1.45, 1.40)1.09 (1.37, 0.81)NAV62K6.08 (5.32, 6.84)0.48 (0.38, 0.57)6.42 (7.44, 5.40)10.0 (11.4, 8.60)0.16 (0.18, 0.14)1.18 (1.13, 1.23)0.37 (0.44, 0.30)NAV62L4.25 (1.46, 7.04)0.59 (0.26, 0.91)59.9 (65.3, 54.4)3.26 (3.58, 2.94)0.61 (0.59, 0.63)1.39 (1.43, 1.35)9.27 (10.2, 8.34)NAV62M29.7 ± 6.291.99 ± 0.3853.6 ± 2.514.88 ± 0.250.46 ± 0.031.49 ± 0.124.68 ± 0.48NAV62Q6.69 (7.04, 6.34)0.61 (0.55, 0.66)14.9 (14.9, 14.8)12.7 (11.5, 13.9)0.11 (0.11, 0.10)1.14 (1.16, 1.12)0.75 (0.75, 0.74)NAV62T14.3 (15.4, 13.2)1.23 (1.13, 1.32)25.7 (26.6, 24.8)26.3 (30.5, 22.1)0.22 (0.31, 0.13)1.36 (1.47, 1.25)0.26 (0.27, 0.24)4.07 (5.85, 2.29)a GI, the activity index for the enzyme was calculated as previously described (24.Christesen H.B. Jacobsen B.B. Odili S. Buettger C. Cuesta-Munoz A. Hansen T. Brusgaard K. Massa O. Magnuson M.A. Shiota C. Matschinsky F.M. Barbetti F. Diabetes. 2002; 51: 1240-1246Crossref PubMed Scopus (138) Google Scholar)b NA, not applicable Open table in a new tab To explain how an enzyme that is kinetically more active than wild type causes MODY we investigated whether the mutation might result in an enzyme that is thermally labile. Analysis of the thermal stability of GST-GCK showed that the wild type enzyme was slightly activated after 30-min incubation as the temperature was raised from 30 to 47.5 °C but that its kcat decreased at higher temperatures falling to about 10 s−1 (by 80%) at 50 °C (Fig. 3a). The activity of V62M GST-GCK increased similarly as that of the wild type as the temperature rose but decreased abruptly at temperatures above 42.5 °C. In contrast, the previously reported instability mutant E300K GST-GCK (16.Gloyn A. Odili S. Buettger C. Njolstad P.R. Shiota C. Magnuson M. Matschinsky F. Matschinsky F.M. Magnuson M.A. Glucokinase and Glycemic Diseases: From the Basics to Novel Therapeutics. 16. Karger, Basel, Switzerland2004: 92-109Google Scholar) showed much greater thermolability than the wild type enzyme and was also more unstable than V62M. The stabilizing effect of glucose on each enzyme, WT, E300K, and V62M GST-GCK was also investigated. The instability of the mutants was tested at 42.5 °C, because this was the highest temperature at which point the least variation between the three enzymes was visible during temperature titration. Decreasing or eliminating of glucose in the heat step had no effect on the activity of wild type GST-GCK, however this treatment markedly lowered the activity (kcat) of the instability mutant E300K from about ∼41 to ∼8 s−1. Glucose removal caused a far less pronounced decrease in the activity of V62M GST-GCK from ∼58 to ∼36 s−1 (Fig. 3b). In fact, glucose titration at 42.5 °C and using physiological glucose levels showed that V62M is virtually as stable as wild type. Inhibition with human and rat GKRP was also determined. WT-GST-GCK showed an expected reduction in activity with increasing concentrations of both human (Fig. 4a) and rat (data not shown) GKRP with and without sorbitol-6-phosphate (S6P). However, there was no reduction in enzyme activity for V62M-GST-GCK with either human (Fig. 4, a and b) or rat GKRP (data not shown). Further kinetic

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