Order–Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase
Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturityâonset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorderâorder transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorderâorder cycle of the small domain as a âtime-delay loop,â which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions. Glucokinase is a key metabolic enzyme that functions as the body's principal glucose sensor. Glucokinase regulates the rate at which insulin is secreted by the pancreas by using a unique but poorly understood cooperative kinetic response to increasing glucose concentrations. The physiological importance of this enzyme is underlined by the fact that mutations in the glucokinase gene lead to maturity-onset diabetes of the young type II (MODY II), permanent neonatal diabetes mellitus (PNDM), and hypoglycemic hyperinsulinemia of infancy (HI). In this study, we use cutting-edge high-resolution nuclear magnetic resonance methods to understand how the kinetic properties of glucokinase contribute to glucose homeostasis. We also seek to understand how a class of recently discovered small-molecule drugs, which hold promise as therapeutics for type 2 diabetes, function to enhance glucokinase activity. Our results suggest that glucokinase samples a range of conformational states in the absence of glucose. However, in the presence of glucose or a small-molecule activator, the enzyme population shifts towards a more narrow, well-structured ensemble of states. Our findings provide a new model for glucokinase cooperative kinetics, which relies on a slow orderâdisorder transition in response to glucose concentrations. These results also reveal a universal mechanism of glucokinase activation, which may inform the development of new antidiabetic agents.