Download ppt

Glucokinase, a glucose sensor
Allison Craney
November 30, 2006
Glucokinase (GK): Biological Significance
• Dramatic conformational change when activated
• Allosteric activator binding
• Monomeric protein which displays cooperativity
• Potential anti-diabetic therapeutic
• Current. Crystal structures solved in 2004
GK: basic properties
• Catalyzes glucose-6-phosphate production
ATP
GK
• Hexokinase family member with important distinctions
– low affinity for glucose
– not regulated by end products
• Responsible for 20% total catalysis of glucose
• 80% in Beta cells (pancreas) and hepatocytes (liver)
Molecules: www.steve.gb.com/science/core_metabolism.html
Glucose homeostasis is tightly regulated
• Mammalian blood glucose level (G): 5mM
G: 7-8mM
G: <5mM
Pancreatic beta cells
produce insulin
Glucose Uptake
Liver alpha cells
produce glucagon
Stimulates
Catabolism
GK is a glucose sensor in Beta cells
• Triggers insulin release via metabolic coupling of the Na+
and Ca++ channels
• Supply-driven pathway
– Low affinity for G
1. G blood level INCREASE
2. ADP increases
GK activity + increased ATP
ATP dependent Na+ channel closes
3. Closing allows the opening of Ca2+ channel, coating cytoplasm with Ca2+
4. Insulin is released
• Activity lowers blood G level
Structure Determination
•Crystallization via hanging drop method
•Structure solved by molecular replacement, using AMORE and CCP4
programs
1V4S
1V4T
Resolution (A)
2.3
3.4
Rworking (%)
23.2
23.8
Rfree (%)
27.4
30.6
Average (A)2
B-factor
~40
39.2
Determinants
Structural properties of GK
•50 kDa, 465 amino acids,
mainly alpha helical
•Smallest hexokinase
–Structurally diverse, but has
conserved actin fold
QuickTime™ and a
Sorenson Video 3 decompressor
are needed to see this picture.
•Monomer, palm-shaped
•Binds G, ATP, activator
1V4S. GK, closed conformation
GK Active Conformation
Deep purple: allosteric activaton molecule
Violet: glucose
Light pink: ATP
PDB ID 1V4S
Glucose binding cleft
PDB ID 1V4S
Allosteric site close-ups:
Activator bound
PDB ID 1V4S
GK undergoes a large conformational change
• Two conformations of GK
– CLOSED (active form). Glucose plus allosteric activator
bound.
– OPEN (inactive form). No glucose bound.
• Between two states, huge rotation of 120 degrees.
• Conversion is much slower than catalytic activity
– Only monomeric protein to display cooperativity
– Hill coefficient = 1.9
Deep blue: flexible loop which covers G binding
Green: Small subunit G binding residues
Teal: Asp205 (large subunit)
Purple: Activator binding residues in allosteric site
PDB ID 1V4T
GK Conformational Change
Closed conformation
PDB ID 1V4S
“Super-open” conformation
PDB ID 1V4T
GK mutations decrease glucose homeostasis
• >200 human GK mutations have been isolated
• (25-30% glycolysis in Beta cells: 5mM blood G)
• Loss-of-function mutations: much more G needed to reach
glycolytic activity level. Elevated blood G.
Condition: early onset diabetes mellitus
• Gain-of-function mutations: less G is needed.
Condition: hypoglycemia. Patients’ blood G: 1.5-3mM.
• Potential antidiabetic drug- screen of >120,000 compounds.
RO-28-1675 decreased blood G while increasing insulin
levels above basal.
QuickTime™ and a
Sorenson Video 3 decompressor
are needed to see this picture.
Selected Literature
•
•
•
•
•
Grimsby, et al. 2003. Allosteric activators of Glucokinase: potential role in diabetes therapy.
Science. 301: 370-373.
Kamata, et al. 2004. Structural basis for allosteric regulation of the monomeric allosteric enzyme
human glucokinase. Structure. 12: 429-438.
Zelent, et al. 2005. Glucokinase and glucose homeostasis: proven concepts and new ideas.
Bioch. Soc. Trans. 33 (1): 306-310.
PDB ID 1V4S and 1V4T (Kamata, et al., 2004) were morphed using Yale Morph Server
All movies and figures were produced in Pymol Molecular Graphics System (DeLano Scientific
LLC, San Carlos, CA; http://www.pymol.org).