1. Lecture #14 Regulatory Enzymes

2. Outline Phosphofructokinase-1 Describing the bound states of activators and inhibitors Integration with glycolysis

3. Phosphofructokinase-1

4. Metabolic Role

5. Background Tetramer 3 Isoforms: M,L,P (muscle, liver, platelet) 2 Natural Forms: R,T (relaxed, tight) Known inhibitors: ATP, citrate, PEP Known activators: AMP, cAMP, Pi, SO 4, FBP Catalytic Activity:

6. PFK sub-network

7. The Catalytic Mechanism: binding of the two substrates followed by the chemical reaction 1) 2) 3)

8. AMP and ATP as regulatory ligands activation inhibition conformation

9. Stoichiometric Matrix

10. Pools and Ratios PFK – R state –All forms of R 0 + R 1 + R 2 + R 3 + R 4 PFK – T state –All forms of T 0 + T 1 + T 2 + T 3 + T 4 PFK – R catalytic state –All forms of R i,AF Ratios At steady state ~ r R = 90%, r cat = 12%

11. DETERMINING THE STEADY STATE

12. Let’s revisit the subnetwork Equilibrium v = 0 Steady State

13. Constraints on the Network Total mass balance: Total flux: Known equilibrium constants

14. Solving for the concentrations Note: When equilibrium constants are plugged in, all forward rate constants in equilibrium reactions fall out, leaving only the catalytic rate constants

15. Estimating the catalytic rate constants Chosen Steady State k PFK k F6P k ATP

16. INTEGRATION WITH GLYCOLYSIS

17. Stoichiometric Matrix

18. DYNAMIC SIMULATIONS

19. Dynamic Simulation Two perturbations –Standard 50% increase in ATP utilization –Additional 15% decrease in ATP utilization

20. Glycolysis Dynamics

21. 50% increase in ATP utilization15% decrease in ATP utilization

22. Summary Enzymes can be explicitly represented in simulation modules as molecules Enzymes have many binding states Binding of regulators (inhibitors and activators) alters protein activity; leading to a ‘tug of war’ amongst the functional states (i.e. T and R) Ratios that represent what fraction of the enzyme is in an active or inhibited functional states can be formed Enzyme sub-networks can be seamlessly integrated with the scaffold metabolic network Regulator binding to PFK, a key glycolytic regulatory enzyme, was demonstrated