Article

Structure/function studies of human beta-cell glucokinase. Enzymatic properties of a sequence polymorphism, mutations associated with diabetes, and other site-directed mutants.

Howard Hughes Medical Institute, University of Chicago, Illinois 60637.
Journal of Biological Chemistry (Impact Factor: 4.6). 07/1993; 268(20):15200-4.
Source: PubMed

ABSTRACT Glucokinase plays a key role in the regulation of glucose metabolism in insulin-secreting pancreatic beta-cells and in the liver. Recent studies have shown that mutations in this enzyme can lead to the development of a form of non-insulin-dependent diabetes mellitus that is characterized by an autosomal dominant mode of inheritance and onset during childhood. Here, we report the catalytic properties of five additional missense mutations associated with diabetes (Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, Trp257-->Arg and Lys414-->Glu), one polymorphism present in both normal and diabetic subjects (Asp4-->Asn), and three site-directed mutations (Glu177-->Lys, Glu256-->Ala, and Lys414-->Ala). The Trp257-->Arg mutation generated an enzyme that had an activity that was less than 0.5% of that for native human beta-cell glucokinase. By contrast, the Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, and Lys414-->Glu mutations had a Vmax that was 20-100% of normal but a Km for glucose that was 8-14-fold greater than the native enzyme. There was no effect of the Asp4-->Asn polymorphism or the Glu177-->Lys substitution on glucokinase activity. The Lys414-->Ala substitution had no effect on Vmax but increased the Km for glucose 2-fold and the Glu256-->Ala substitution caused a approximately 200-fold decrease in Vmax. These studies have led to the identification of additional residues involved in glucokinase catalysis and substrate binding.

Download full-text

Full-text

Available from: Philippe Froguel, Jul 03, 2015
0 Followers
 · 
60 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study the glucose responsiveness of isolated, overnight-cultured islets of obese LA/N-corpulent (cp/cp) rats was compared with glucose phosphorylating activity to determine whether changes in the function of glucokinase could be identified. Islets from both male and female cp/cp rats showed a left-shifted concentration response to glucose, with EC50 values of 1.5 and 4.6 mM, respectively, compared with 9.2 mM for lean control islets. Islets from cp/cp rats were partially resistant to inhibition by mannoheptulose, a glucokinase inhibitor. Minimum inhibitory concentrations were 10 mM in cp/cp vs. 3 mM in lean rat islets. Glucose phosphorylating potential was markedly increased in islets of male cp/cp, but not female cp/cp, compared with lean rats. The maximal velocity (Vmax) of hexokinase was increased 5-fold, while the Km of glucokinase was significantly decreased, in male cp/cp compared with the lean control islets(3.6 vs. 35.2 mM). The Km for glucokinase was also decreased in female cp/cp rat islets (17.2 mM). The data from male cp/cp rat islets are consistent with the idea that increased glucose phosphorylation capacity can contribute to insulin hypersecretion and an extreme leftward shift in the concentration-response curve. However, other factors must also be considered because female cp/cp rats have moderately increased insulin secretory capacity without marked changes in total glucose phosphorylating capacity.
    Canadian Journal of Physiology and Pharmacology 05/1995; 73(4):501-8. DOI:10.1139/y95-063 · 1.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorylation of glucose to glucose 6-phosphate by glucokinase (GK; EC 2.7.1.2) serves as a glucose-sensing mechanism for regulating insulin secretion in beta cells. Recent findings of heterozygous GK gene mutations in patients with maturity-onset diabetes of the young (MODY), a form of type II (non-insulin-dependent) diabetes characterized by autosomal dominant inheritance, have raised the possibility that a decrease in beta-cell GK activity may impair the insulin secretory response of these cells to glucose. To generate an animal model for MODY we have expressed in transgenic mice a GK antisense RNA with a ribozyme element under control of the insulin promoter. Mice in two independent lineages had about 30% of the normal islet GK activity. Insulin release in response to glucose from in situ-perfused pancreas was impaired; however, the plasma glucose and insulin levels of the mice remained normal. These mice are likely to be predisposed to type II diabetes and may manifest increased susceptibility to genetic and environmental diabetogenic factors. They provide an animal model for studying the interaction of such factors with the reduced islet GK activity.
    Proceedings of the National Academy of Sciences 04/1994; 91(6):2051-5. DOI:10.1073/pnas.91.6.2051 · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In eight glucokinase (GCK)-deficient subjects, we have investigated insulin secretion rates (ISRs) in response to intravenous arginine. Impairment in the enzymatic activity of mutant GCK leads to a reduced glycolytic flux in beta-cells. This defect translates in vivo as a right shift in the glucose/SR dose-response curve. Insulin secretion in response to other secretagogues has not been reported. The arginine test was performed immediately after a 2-h hyperglycemic (10 mM) clamp. ISR was computed by deconvolution of peripheral C-peptide levels. Linear regression analyses were performed to assess correlations between the beta-cell secretory responses to the arginine test, an intravenous glucose tolerance test (IVGTT), and a hyperglycemic clamp (areas under the C-peptide curves), and between these parameters and the glucose tolerance status (area under the glucose curve during an oral glucose tolerance test). Two minutes after the injection of arginine, the increment in ISR was 30.17 +/- 10.01 pmol insulin.kg-1.min-1 in patients and 36.25 +/- 15.46 pmol insulin.kg-1.min-1 in control subjects (P = 0.38). Throughout the experiment, increments in ISR were comparable in both groups. The amount of insulin secreted in response to arginine (0-5 min) was similar in patients and control subjects: 81 +/- 28 vs. 119 +/- 55 pmol/kg (P = 0.16), respectively. The arginine test C-peptide response was not correlated with the IVGTT or hyperglycemic clamp responses. The arginine test and hyperglycemic clamp responses were not correlated to the glucose tolerance status. The best predictor of the glucose tolerance was the C-peptide response to the IVGTT (r2 = 0.78; P = 0.002). beta-cell secretory increment in response to arginine was found to be in the normal range in GCK-deficient subjects. The arginine test does not seem to reflect either the beta-cell secretory defect or the glucose tolerance status of these subjects. IVGTT seems to be the best predictor of the latter parameter in this population.
    Diabetes Care 10/1994; 17(9):1015-21. DOI:10.2337/diacare.17.9.1015 · 8.57 Impact Factor