Glucose metabolism in humans is tightly controlled by the activity of glucokinase (GCK). GCK is predominantly produced in the pancreas, where it catalyzes the rate-limiting step of insulin secretion, and in hepatocytes, where it participates in glycogen synthesis. A multitude of diseasecausing mutations within the gck gene have been identified. Activating mutations manifest themselves in the clinic as congenital hyperinsulinism, while loss-of-function mutations produce several diabetic conditions. Indeed, pharmaceutical companies have shown great interest in developing GCK-associated treatments for diabetic patients. Due to its essential role in maintaining whole-body glucose homeostasis, GCK activity is extensively regulated at multiple levels. GCK possesses a unique ability to self-regulate its own activity via slow conformational dynamics, which allows for a cooperative response to glucose. GCK is also subject to a number of protein-protein interactions and post-translational modification events that produce a broad range of physiological consequences. While significant advances in our understanding of these individual regulatory mechanisms have been recently achieved, how these strategies are integrated and coordinated within the cell is less clear. This review serves to synthesize the relevant findings and offer insights into the connections between molecular and cellular control of GCK.
Fresh water cyanobacterial algal blooms represent a major health risk because these organisms produce cylindrospermopsin, a toxic, structurally complex, zwitterionic uracil-guanidine alkaloid recognized by the EPA as a dangerous drinking water contaminant. At present, the ability to detect and quantify the presence of cylindrospermospin in water samples is severely hampered by the lack of an isotopically labeled standard for analytical mass spectrometry. Herein, we present a concise, scaled total synthesis of N cylindrospermosin fromN ammonium chloride, which leverages a unique stereoselective intramolecular double conjugate addition step to assemble the tricyclic guanidine core. In addition to providing the first pure isotopically labeled probe for precise quantification of this potent biotoxin in fresh water sources, our results demonstrate how unique constraints associated with isotope incorporation compel novel solutions to synthesis design.
Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a β-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the β-type variant lacks a burst phase. Comparative hydrogen-deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active-site loop, which folds upon glucose binding, is protected from exchange in the α-type variant. The α-type variant also displays increased exchange within a β-strand located near the enzyme’s hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, β-type activation causes no substantial difference in global or local exchange relative to unliganded, wild-type GCK. Together these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas β-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK’s unique kinetic cooperativity.
Despite their biological importance, post‐translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur‐based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ‐selenolysine, a selenol‐containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ‐selenolysine is demonstrated with the traceless ligation of the small ubiquitin‐like modifier protein, SUMO‐1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin‐like modifying protein to its target.
Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecondtimescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19 F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.
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