Journal Article

Order–Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase

Miller, Brian G., Larion, Mioara, Brüschweiler, Rafael, Salinas, Roberto Kopke, Bruschweiler-Li, Lei

2012

Overview

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 maturity-onset 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 disorder-order 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 disorder-order cycle of the small domain as a \"time-delay loop,\" which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Allosteric Regulation / Amino Acid Substitution - genetics / Biology / Blood sugar / Catalytic Domain / Congenital Hyperinsulinism - drug therapy / Congenital Hyperinsulinism - enzymology / Congenital Hyperinsulinism - genetics / Diabetes / Enzyme Activation / Enzyme Stability / Enzymes / Glucokinase - chemistry / Glucokinase - metabolism / Glucose / Glucose - metabolism / Health aspects / Humans / Isoleucine - chemistry / Kinetics / Magnetic Resonance Spectroscopy / Models, Biological / Models, Molecular / NMR / Nuclear magnetic resonance / Pancreas / Phosphotransferases / Physiological aspects / Protein Structure, Secondary / Protein Structure, Tertiary