Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 112, Issue 37, Pages 11553-11558Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1506664112
Keywords
allostery; monomeric cooperativity; glucokinase; diabetes; intrinsic disorder
Categories
Funding
- American Heart Association [12POST12040344]
- National Science Foundation [MCB-0918362]
- NIH [1R01DK081358]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1360966] Funding Source: National Science Foundation
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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 a-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the H-1-C-13 heteronuclear multiple quantum coherence (HMQC) spectrum of C-13-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 H-1-C-13 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.
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