The hexokinase (HK) family of enzymes catalyze the conversion of glucose to glucose 6-phosphate in a process that is utilized for energy production in most organisms. Hexokinase IV, commonly known as glucokinase (GCK), is functionally distinct from the rest of the HK isozymes. It is characterized by a high substrate concentration at half-maximal velocity and is not inhibited by an abundance of its product. GCK also displays positive kinetic cooperativity, despite functioning as a monomer and containing only one glucose binding site. This responsiveness is such that the inflection point occurs in the range of physiological blood glucose levels, providing the enzyme with exceptional sensitivity in this region. GCK's unique functional properties allow it to control the rates of insulin release and glycogen synthesis. The significance of proper GCK function is emphasized by various pathological conditions that arise from mutations in the gck gene. These discoveries are expanded upon in Chapter 1 and have led to GCK being considered the glucose sensor of the body. Millisecond timescale fluctuations of the small domain have been shown to be essential for cooperativity in GCK. However, a detailed picture of GCK's dynamic conformational landscape, including the number of accessible states, their relative populations, and the timescales on which they interconvert is absent in the literature. In Chapter 2, we map the intrinsic dynamics and structural heterogeneity of GCK on the nanosecond timescale using a combination of unnatural amino acid incorporation, time-resolved fluorescence spectroscopy and 19F nuclear magnetic resonance spectroscopy. Based on these results, we propose a catalytic model in which cooperativity originates from correlation between nanosecond and millisecond timescale motions. Activating GCK mutations abolish cooperativity and manifest themselves in the clinic as congenital hyperinsulinism. In Chapter 3, we use steady- and transient-state kinetics, and hydrogen-deuterium exchange mass spectrometry, to demonstrate that mutational activation of GCK occurs via two distinct mechanisms: α and β. Our data reveal that α-activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound, closed conformation. β-type activation is instead caused by increased mobile loop dynamics, which accelerate the product release rate. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants. Due to its essential role in maintaining whole-body glucose homeostasis, GCK activity is extensively regulated at virtually every level in the cell. The hormonal, metabolic, and transcriptional regulation of GCK have been described in great detail by other laboratories.1,2 Protein-protein interactions and post-translational modifications involving GCK elicit an array of physiological consequences and intrinsic conformational dynamics provide GCK with an additional layer of functional control. In Chapter 4, we offer insights into how these regulatory strategies are integrated and coordinated within the broader context of the cell. Of these regulatory mechanisms, the post-translational conjugation of the small ubiquitin-like modifier (SUMO1) protein to GCK remains one of the most poorly understood. Recently, it was reported that SUMOylation increases GCK's activity and stability, and mediates nuclear translocation of the enzyme.3,4 However, the inability to isolate homogenous, SUMOylated proteins often inhibits full characterization of the modification. In Chapter 5 we describe our efforts to generate SUMOylated GCK using semi-synthetic and coexpression approaches. We conclude with a look to the future, emphasizing the need for continued investigation and describing future experiments. In Chapter 6, we deviate from investigations of GCK and describe our efforts to characterize CyrI, a unique iron-dependent, nonheme oxygenase. This enzyme is expressed in cyanobacteria, where it catalyzes the final step in the biosynthesis of the toxic drinking water contaminant cylindrospermopsin. CyrI catalyzes a challenging C-H oxidation step with exquisite selectivity and appears to be depend on a sulfate group as a substrate recognition motif. CyrI is intriguing to develop from a chemical synthesis perspective as the selective functionalization of C-H bonds among numerous similarly reactive C-H bonds is a considerable challenge in organic synthesis. We detail our analysis of CyrI stability and crystallization and provide insights into future experimentation.
