Abstract
Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing.
Highlights
Glucokinase (GK) plays a key role as the pancreatic beta-cell glucose sensor by integrating blood glucose levels and glucose metabolism with insulin secretion [1]
In this report we describe the functional analysis of five GCK mutations identified in probands with familial, mild fasting hyperglycaemia
The structural data obtained from the free and substrate/ activator bound GK crystals showed that this enzyme has a small and a large domain separated by a deep cleft where substrates bind [18]
Summary
Glucokinase (GK) plays a key role as the pancreatic beta-cell glucose sensor by integrating blood glucose levels and glucose metabolism with insulin secretion [1]. Besides the beta-cell, GK is expressed in other neuroendocrine cells and in hepatocytes, which participate in a network of cells involved in the body glucose homeostasis [2]. This enzyme ( called hexokinase IV) is one of the four hexokinase isoforms that catalyze the first reaction of glycolysis converting glucose into glucose-6-phosphate (G6P) with ATP as second substrate. The best known regulator of GK in the liver is the glucokinase regulatory protein (GKRP) that acts as a competitive inhibitor with respect to glucose and regulates the nucleo-cytoplasmic localization of the enzyme to stabilize a protein reservoir of the enzyme in the nucleus when its activity is not necessary [6] [7] [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
