期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：37
页码：11553-11558
DOI：10.1073/pnas.1506664112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceGlucokinase (GCK), the body's primary glucose sensor, displays a unique sigmoidal kinetic response that is a hallmark of allostery. Allostery in GCK is different from textbook models, because the enzyme is monomeric and contains only one glucose binding site. Previously, synthetic activators and activating disease mutations were thought to share a common mechanism of allosteric activation in which GCK is shifted toward a glucose-bound-like state. Using mutagenesis and genetic selection, we identify an activated variant that utilizes a different mechanism. Limited proteolysis and NMR reveal that activation is achieved by modulating the dynamic properties of an active-site loop without perturbing the ensemble structure. This previously undescribed activation mechanism is shown to operate in naturally occurring, hyperinsulinemia-associated disease variants. Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In -type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile-labeled enzyme resembles the glucose-bound state. In {beta}-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.
