Characterizing glucokinase variant function and mechanisms

Whole genome sequencing of patients is increasingly feasible, facilitating accurate and fast diagnosis. Genetic diagnosis, however, also requires variant interpretation. Although the effects of some variants are known, most variants are of unknown significance, hindering genetic diagnosis. The focus in this thesis is on how variants impact the human enzyme glucokinase (GCK). GCK functions as a glucose sensor, as GCK activity regulates glycogen synthesis and insulin secretion. Accordingly, variants that perturb GCK function are linked to glucose homeostasis diseases. In the two studies presented in this thesis, we examine GCK variant effects and mechanisms experimentally and computationally. In the first study, we multiplexed a yeast functional complementation assay to characterize the function of nearly all GCK missense variants, including both variants with increased and decreased function. We found that hyperactive variants were concentrated at an allosteric activator site, while hypoactive variants were found at buried residues including the active site. In addition, we examined variant effects mechanistically using predicted changes in protein thermodynamic stability for the active and inactive conformations of GCK. To further explore GCK variant mechanisms, we, in the second study, assayed GCK variant abundance. We found that abundance was mainly decreased by variants in buried residues in the large domain. Finally, we extended our previous stability predictions, and found that the mechanism of variants in the small domain was mainly to perturb protein conformational dynamics. Collectively, the two studies presented in this thesis characterize the effects and mechanisms of GCK variants. The results may be useful for variant interpretation, and thus diagnosis of GCK-linked diseases. The main impact, however, is an increased understanding of the intriguing enzyme GCK, which may in turn facilitate the development of therapeutics for both GCK-linked diseases and type 2 diabetes.