Lecture #14

Regulatory Enzymes

Outline

• Phosphofructokinase-1• Describing the bound states of

activators and inhibitors• Integration with glycolysis

Phosphofructokinase-1

Metabolic Role

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, SO4, FBP• Catalytic Activity:

PFK sub-network

The Catalytic Mechanism:binding of the two substrates followed by the

chemical reaction

1)

2)

3)

AMP and ATP as regulatory ligands

activation

inhibition

conformation

Stoichiometric Matrix

Pools and Ratios• PFK – R state

– All forms of R0 + R1 + R2 + R3 + R4

• PFK – T state– All forms of T0 + T1 + T2 + T3 + T4

• PFK – R catalytic state– All forms of Ri,AF

• Ratios

• At steady state ~ rR = 90%, rcat = 12%

DETERMINING THE STEADY STATE

Let’s revisit the subnetwork

Equilibrium v = 0

Steady State

Constraints on the Network

• Total mass balance:

• Total flux:

• Known equilibrium constants

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

Estimating the catalytic rate constants Chosen Steady State

kPFK

kF6P

kATP

INTEGRATION WITH GLYCOLYSIS

Stoichiometric Matrix

DYNAMIC SIMULATIONS

Dynamic Simulation

• Two perturbations– Standard 50% increase in ATP utilization– Additional 15% decrease in ATP utilization

Glycolysis Dynamics

Glycolysis Dynamics

50% increase in ATP utilization 15% decrease in ATP utilization

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