Dual allosteric activation mechanisms in monomeric human glucokinase.
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 α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C 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.
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306;
A Carl
AC
Whittington
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210;
Mioara
M
Larion
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306;
Joseph M
JM
Bowler
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093.
Kristen M
KM
Ramsey
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210;
Rafael
R
Brüschweiler
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306; miller@chem.fsu.edu.
Brian G
BG
Miller
D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000235:genetics; D015723:Gene Library; D005941:Glucokinase / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry; D006593:Hexokinase / Q000737:chemistry; D006801:Humans; D006946:Hyperinsulinism / Q000235:genetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D016296:Mutagenesis; D009154:Mutation; D017433:Protein Structure, Secondary
D008024:Ligands; D006593:Hexokinase; D005941:Glucokinase; D005947:Glucose
allostery; diabetes; glucokinase; intrinsic disorder; monomeric cooperativity