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Allosteric Activation of Human Glucokinase

Title: Allosteric Activation of Human Glucokinase.
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Name(s): Bowler, Joseph M., author
Miller, Brian G., professor directing dissertation
Stefanovic, Branko, university representative
Logan, Timothy M., 1961-, committee member
Yang, Wei, committee member
Zhu, Lei, 1978-, committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Department of Chemistry and Biochemistry, degree granting department
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2014
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (137 pages)
Language(s): English
Abstract/Description: The hexokinase family of enzymes catalyzes the ATP-dependent phosphorylation of glucose to generate glucose 6-phosphate and ADP. Consistent with highly evolved catalysts, hexokinases I-III possess high apparent substrate affinities (low Km) as a result of specificity optimization. Hexokinase IV, commonly known as glucokinase (GCK), has a low apparent affinity for its physiological substrate glucose and is not significantly inhibited by the reaction product. Interestingly, it displays positively cooperative kinetic behavior while functioning exclusively as a monomer with a single glucose binding site. A lag in steady-state saturation curves places the inflection point in the range of physiological blood glucose levels, allowing activity to be highly tunable in this region. Fasting and fed states can therefore dictate the rate of glucose phosphorylation in the reaction that represents the rate-limiting factor of insulin release and glycogen synthesis. The most compelling evidence for the pivotal role of GCK in glucose homeostasis was the discovery of mutations in the glk gene that result in long-term glycemic complications. Established as a central regulator of blood glucose, efforts to target GCK in the treatment of diabetic disorders resulted in the development of glucokinase activators. Small-molecule activators of GCK bind to an allosteric site and increase the enzyme's apparent affinity for glucose (K0.5). Effects on turnover (kcat) and cooperativity (Hill coefficient) can vary widely, and these differences have remained largely uninvestigated. Conflicting kinetic models have been proposed in the literature that disagree as to whether glucose binding is pre-requisite to activator association. Our kinetic activity assays, obtained via stopped-flow, suggest that activators can indeed associate with some population of GCK in the absence of glucose. To investigate the differing effects on turnover by activators, we performed viscosity variation assays. We found that the kcat of GCK is partly dependent on some diffusion-limited process, such as product release. In the presence of different activators, whose effects on turnover varied from unchanged to augmented 30%, we observed a significant change in dependence for just one of the three compounds tested. Compound A increased kcat by 10% and showed a nearly complete dependence upon solvent viscosity, suggesting that the activator functions by accelerating some preceding event that makes product release the determinant for turnover. The precise in vivo regulation of GCK and the presence of its highly effective allosteric activation site have led some to speculate the existence of an endogenous activator. Such a molecule would likely provide a safe scaffold in the design of future GCK therapeutics. We tested a variety of biogenic compounds using linked-enzyme assays for activating properties. When these tests proved unsuccessful, we developed a methodology that would provide the ability to genetically select for activating cyclic peptides among a library of 106 unique members. We combined a proven genetic selection system devised in-house with a uniquely engineered plasmid construct that generates randomized cyclic peptides in vivo. The methodology was optimized to be sensitive to the presence of GCK activation. In addition, we describe alternative approaches and ongoing efforts on this front.
Identifier: FSU_migr_etd-9147 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Fall Semester, 2014.
Date of Defense: November 4, 2014.
Keywords: activation, glucokinase
Bibliography Note: Includes bibliographical references.
Advisory Committee: Brian Miller, Professor Directing Dissertation; Branko Stefanovic, University Representative; Timothy Logan, Committee Member; Wei Yang, Committee Member; Lei Zhu, Committee Member.
Subject(s): Biochemistry
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_migr_etd-9147
Owner Institution: FSU

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Bowler, J. M. (2014). Allosteric Activation of Human Glucokinase. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-9147