1. Glucokinase, a glucose sensor Allison Craney November 30, 2006

2. 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

3. GK: basic properties Catalyzes glucose-6-phosphate production Hexokinase family member with important distinctions –low affinity for glucose –not regulated by end products Responsible for 20% total catalysis of glucose 80%80% in Beta cells (pancreas) and hepatocytes (liver) ATPGK Molecules: www.steve.gb.com/science/core_metabolism.html

4. Glucose homeostasis is tightly regulated Mammalian blood glucose level (G): 5mM G: 7-8mM Pancreatic beta cells produce insulin Glucose UptakeLiver alpha cells produce glucagon Stimulates Catabolism G: <5mM

5. 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 Activity lowers blood G level 1. G blood level INCREASE GK activity + increased ATP 2. ADP increases ATP dependent Na+ channel closes 3. Closing allows the opening of Ca2+ channel, coating cytoplasm with Ca2+ 4. Insulin is released

6. Structure Determination Determinants 1V4S1V4T Resolution (  A) 2.33.4 R working (%)23.223.8 R free (%)27.430.6 Average (A)  2 B-factor ~4039.2 Crystallization via hanging drop method Structure solved by molecular replacement, using AMORE and CCP4 programs

7. Structural properties of GK 50 kDa, 465 amino acids, mainly alpha helical Smallest hexokinase –Structurally diverse, but has conserved actin fold Monomer, palm-shaped Binds G, ATP, activator 1V4S. GK, closed conformation

8. Deep purple: allosteric activaton molecule Violet: glucose Light pink: ATP GK Active Conformation PDB ID 1V4S

9. Glucose binding cleft PDB ID 1V4S

10. Allosteric site close-ups: Activator bound PDB ID 1V4S

11. 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

12. 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

13. GK Conformational Change PDB ID 1V4SPDB ID 1V4T “Super-open” conformation Closed conformation

14. 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.

16. 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).