Glucokinase is a key regulator of glucose homeostasis in the human body. Dysfunction in the regulation and/or activity of glucokinase causes a diverse range of diseases including maturity onset diabetes of the young (MODY) and hyperinsulinemia of infancy (HI). Human glucokinase is a monomeric enzyme that is allosterically regulated by its sugar substrate, D- glucose. This kinetic cooperativity is critical to maintaining proper glucose serum levels in vivo. The goal of this study is to elucidate the mechanistic basis for cooperativity in human glucokinase. The experiments described herein combine a powerful genetic selection system for human glucokinase activity with mechanistic enzymology to provide a kinetic and molecular description of the conformational transitions that give rise to cooperativity.
The specific aims are: (1) To determine the mechanism of activation of hyperinsulinemia-linked glk mutations;(2) To investigate conformational heterogeneity in unliganded glucokinase via chemical quench-flow techniques;(3) To investigate the role of a secondary structural """"""""allosteric switch"""""""" in glucokinase cooperativity;(4) To determine whether prolyl cis/trans isomerization is responsible for the slow interconversion of enzyme conformations. Achieving the objectives outlined in this application promises to impact the development of future glucokinase-targeted therapeutics for diabetes and hyperinsulinemia.

Public Health Relevance

In the past five years, human glucokinase has emerged as an attractive therapeutic target for diabetes. Numerous small-molecule activators of glucokinase have been identified and several have been shown to be effective at modulating blood glucose levels in animal models. The successful completion of the aims outlined in this application will provide critical new information about the mechanisms of glucokinase regulation in vivo. This information has the potential to significantly impact future efforts to design glucokinase-targeted therapeutics for diabetes and hyperinsulinemia.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Sechi, Salvatore
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Florida State University
Schools of Arts and Sciences
United States
Zip Code
Sternisha, Shawn M; Liu, Peilu; Marshall, Alan G et al. (2018) Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism. Biochemistry 57:1632-1639
Rexford, Alix; Zorio, Diego A R; Miller, Brian G (2017) Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD. PLoS One 12:e0171587
Martinez, Juliana A; Xiao, Qing; Zakarian, Armen et al. (2017) Antidiabetic Disruptors of the Glucokinase-Glucokinase Regulatory Protein Complex Reorganize a Coulombic Interface. Biochemistry 56:3150-3157
Reno, Candace M; Puente, Erwin C; Sheng, Zhenyu et al. (2017) Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation. Diabetes 66:587-597
Casey, Ashley K; Miller, Brian G (2016) Kinetic Basis of Carbohydrate-Mediated Inhibition of Human Glucokinase by the Glucokinase Regulatory Protein. Biochemistry 55:2899-902
Whittington, A Carl; Larion, Mioara; Bowler, Joseph M et al. (2015) Dual allosteric activation mechanisms in monomeric human glucokinase. Proc Natl Acad Sci U S A 112:11553-8
Larion, Mioara; Hansen, Alexandar L; Zhang, Fengli et al. (2015) Kinetic Cooperativity in Human Pancreatic Glucokinase Originates from Millisecond Dynamics of the Small Domain. Angew Chem Int Ed Engl 54:8129-32
Schulenburg, Cindy; Miller, Brian G (2014) Enzyme recruitment and its role in metabolic expansion. Biochemistry 53:836-45
Martinez, Juliana A; Larion, Mioara; Conejo, Maria S et al. (2014) Role of connecting loop I in catalysis and allosteric regulation of human glucokinase. Protein Sci 23:915-22
Fioramonti, Xavier; Deak, Adam; Deshpande, Srinidhi et al. (2013) Hypothalamic S-nitrosylation contributes to the counter-regulatory response impairment following recurrent hypoglycemia. PLoS One 8:e68709

Showing the most recent 10 out of 23 publications