The long-term goal of research in the Miller lab is to identify and characterize unique enzyme regulatory mechanisms. In this proposal, we investigate two mechanisms that regulate the activity of human glucokinase (GK), a protein that plays a key role in glucose homeostatic maintenance. Dysfunction in GK regulation causes maturity onset diabetes of the young type 2 and hyperlipidemia. The objective of Aim 1 is to elucidate the molecular origins of kinetic cooperativity in monomeric GK. GK cooperativity is fundamentally different from textbook models; it requires neither multiple ligand binding sites nor protein oligomerization. Instead, GK cooperativity results from hysteresis - it displays a time-dependent change in activity in response to changing glucose concentration that is driven by slow conformational changes. In preliminary studies, we have developed a working model for the hysteretic transitions that facilitate GK cooperativity. In our model, unliganded GK undergoes millisecond timescale conformational exchange between a crystallographically observed ground state (E) and an invisible excited state (E*). We have shown that the rate constant for conformational exchange (kex) is comparable in magnitude to the rate constant for turnover (kcat). The equivalency in these rate constants means that glucose binding does not reach equilibrium during the catalytic cycle, which is required for hysteresis to produce monomeric cooperativity.
In Aim 1, we will develop a comprehensive and complete understanding of GK cooperativity by providing a structural description of the E* state and by establishing the mechanism(s) by which cooperativity is abolished in disease variants. The objective of Aim 2 is to use a combination of unnatural amino acid mutagenesis, stopped-flow spectrometry, crystallography and computational simulations to elucidate the mechanistic basis for GKRP-mediated regulation of GK. GKRP acts as a competitive inhibitor of glucose binding to GK. GKRP dysfunction is associated with cardiovascular disease and it has recently emerged as a viable diabetes therapeutic target.
In Aim 2, we seek to understand how the association of different phosphorylated sugars with GKRP modulates affinity toward GK. We will also elucidate the kinetic mechanism of action of newly described drugs that disrupt the GK-GKRP complex. This proposal is significant to human disease because understanding the structural and dynamic basis of GK cooperativity and GKRP-mediated regulation is essential for the development of therapeutic agents that target both proteins. This work is significant to fundamental biochemistry because the structural and dynamic origins of hysteresis are unknown, despite the fact that regulation by hysteretic behavior is well established in enzymology. This proposal is innovative because it involves a unique combination of biophysical methods cutting-edge labeling methods to elucidate the mechanistic origins of kinetic cooperativity in a monomeric, single-site enzyme and to establish the mechanism of GK regulation by a protein-protein interaction.

Public Health Relevance

The proposed research is relevant to public health because glucokinase is the body's principal glucose sensor, and dysfunction in glucokinase regulation causes several glucose homeostatic diseases, including maturity onset diabetes of the young type 2 and persistent hyperinsulinemia of infancy. The development of new diabetes therapeutic agents that target glucokinase and the glucokinase regulatory protein requires a mechanistic understanding of the unique dynamic and allosteric properties of these proteins. For this reason, the proposed research is relevant to the NIH's mission of developing fundamental knowledge that enhances human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM115388-02S1
Application #
9449986
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
2016-06-01
Project End
2020-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
2
Fiscal Year
2017
Total Cost
$47,181
Indirect Cost
$13,004
Name
Florida State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
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Mailyan, Artur K; Chen, Joanna L; Li, Weiwei et al. (2018) Short Total Synthesis of [15N5]-Cylindrospermopsins from 15NH4Cl Enables Precise Quantification of Freshwater Cyanobacterial Contamination. J Am Chem Soc 140:6027-6032
Paukovich, Natasia; Xue, Mengjun; Elder, James R et al. (2018) Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding. J Mol Biol 430:3234-3250
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