Summary
Human beta-cell glucokinase and its liver counterpart displayed a half-saturating concentration of glucose (S0.5) of about 8 mmol/l and a Hill coefficient of 1.7, and were as sensitive to inhibition by the rat liver regulatory protein as the rat liver enzyme. These results indicate that the N-terminal region of glucokinase, which differs among these three enzymes, is not implicated in the recognition of the regulatory protein. They also suggest that the regulatory protein, or a related protein, could modulate the affinity of glucokinase for glucose in beta cells. We have also tested the effect of several mutations, many of which are implicated in maturity onset diabetes of the young. The mutations affected the affinity for glucose and for the regulatory protein to different degrees, indicating that the binding site for these molecules is different. An Asp158 Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affinity for the regulatory protein. The mutations that were found to decrease the affinity for the regulatory protein (Asn166 Arg, Val203 Ala, Asn204 Gln, Lys414 Ala) clustered in the hinge region of glucokinase and nearby in the large and small domains. These results are in agreement with the concept that part of the binding site for the regulatory protein is situated in the hinge region of this enzyme.
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Abbreviations
- S0.5 :
-
Half-saturating concentration of substrate
- MODY:
-
maturity onset diabetes of the young
- PCR:
-
polymerase chain reaction
References
Van Schaftingen E (1989) A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate. Eur J Biochem 179: 179–184
Van Schaftingen E, Detheux M, Veiga-da-Cunha M (1994) Short-term control of glucokinase activity: role of a regulatory protein. FASEB J 8: 414–419
Vandercammen A, Detheux M, Van Schaftingen E (1992) Binding of sorbitol 6-phosphate and of fructose 1-phosphate to the regulatory protein of liver glucokinase. Biochem J 286: 253–256
Vandercammen A, Van Schaftingen E (1991) Competitive inhibition of liver glucokinase by its regulatory protein. Eur J Biochem 200: 545–551
Vandercammen A, Van Schaftingen E (1993) Species and tissue distribution of the regulatory protein of glucokinase. Biochem J 294: 551–556
Malaisse WJ, Malaisse-Lagae F, Davies DR, Vandercammen A, Van Schaftingen E (1990) Regulation of glucokinase by a fructose-1-phosphate-sensitive protein in pancreatic islets. Eur J Biochem 190: 539–545
Andreone TL, Printz RL, Pilkis SJ, Magnuson MA, Granner DK (1989) The amino acid sequence of rat liver glucokinase deduced from cloned cDNA. J Biol Chem 264: 363–369
Magnuson MA, Shelton KD (1989) An alternate promoter in the glucokinase gene is active in the pancreatic β cell. J Biol Chem 264: 15936–15942
Gidh-Jain M, Takeda J, Xu LZ et al. (1993) Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships. Proc Natl Acad Sci USA 90: 1932–1936
Takeda J, Gidh-Jain M, Xu LZ et al. (1993) Structure/function studies of human β-cell glucokinase. Enzymatic properties of a sequence polymorphism, mutations associated with diabetes and other site-directed mutants. J Biol Chem 268: 15200–15204
Xu LZ, Zhang W, Weber IT, Harrison RW, Pilkis SJ (1994) Site-directed mutagenesis studies on the determinants of sugar specificity and cooperative behavior of human β-cell glucokinase. J Biol Chem 269: 27458–27465
Xu LZ, Harrison RW, Weber IT, Pilkis SJ (1995) Human β-cell glucokinase. Dual role of Ser-151 in catalysis and hexose affinity. J Biol Chem 270: 9939–9946
Liang Y, Kesavan P, Wang LQ et al. (1995) Variable effects of maturity-onset-diabetes of youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme. Biochem J 309: 167–173
Nishi S, Stoffel M, Xiang K, Shows TB, Bell GI, Takeda J (1992) Human pancreatic beta-cell glucokinase: cDNA sequence and localization of the polymorphic gene to chromosome 7, band p 13. Diabetologia 35: 743–747
Korany LI, Tanizawa Y, Welling CM, Rabin DU, Permutt MA (1992) Human islet glucokinase gene. Diabetes 41: 807–811
Vandercammen A, Van Schaftingen E (1990) The mechanism by which rat liver glucokinase is inhibited by the regulatory protein. Eur J Biochem 191: 483–489
Studier FW, Moffat BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189: 113–130
Veiga-da-Cunha M, Courtois S, Michel A, Gosselain E, Van Schaftingen E (1996) Amino acid conservation in animal glucokinases. Identification of the residues implicated in the interaction with the regulatory protein. J Biol Chem 271: 6292–6297
Lange AJ, Xu LZ, Van Poelwijk F, Lin K, Granner DK, Pilkis SJ (1991) Expression and site-directed mutagenesis of hepatic glucokinase. Biochem J 277: 159–163
Pilkis SJ, Weber IT, Harrison RW, Bell GI (1994) Glucokinase: structural analysis of a protein involved in susceptibility to diabetes. J Biol Chem 35: 21925–21928
Cardenas ML (1995) Kinetic and structural properties of hepatic hexokinase D, a monomeric cooperative enzyme. Glucokinase: its regulation and role in liver metabolism. Springer-Verlag, Heidelberg, Germany, pp 41–80
Meglasson MD, Matschinsky FM (1986) Pancreatic islet glucose metabolism and regulation of insulin secretion. Diab Metab Rev 2: 163–214
Matschinsky F, Liang Y, Kesavan P et al. (1993) Glucokinase as pancreatic β-cell glucose sensor and diabetes gene. J Clin Invest 92: 2092–2098
Randle PJ (1993) Glucokinase and candidate genes for type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 36: 269–275
Bell GI, Froguel P, Nishi S et al. (1993) Mutations of the human glucokinase gene and diabetes mellitus. Trends Endocrinol Metab 4: 86–90
Bell GI, Pilkis SJ, Weber IT, Polonsky KS (1996) Glucokinase mutations, insulin secretion, and diabetes mellitus. Ann Rev Physiol 58: 171–186
Becker TC, Noel RJ, Johnson JH et al. (1996) Differential effects of overexpressed glucokinase and hexokinase I in isolated islets. J Biol Chem 27: 390–394
Sturis J, Kurland IJ, Byrne MM et al. (1994) Compensation in pancreatic β-cell function in subjects with glucokinase mutations. Diabetes 43: 718–723
Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24: 946–950
Charles RS, Harrison RW, Bell GI, Pilkis SJ, Weber IT (1994) Molecular model of human β-cell glucokinase built by analogy to the crystal structure of yeast hexokinase B. Diabetes 43: 718–723
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Veiga-da-Cunha, M., Xu, L.Z., Lee, Y.H. et al. Effect of mutations on the sensitivity of human beta-cell glucokinase to liver regulatory protein. Diabetologia 39, 1173–1179 (1996). https://doi.org/10.1007/BF02658503
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DOI: https://doi.org/10.1007/BF02658503