1. Lecture 14: Regulation of Proteins 1: Allosteric Control of ATCase
Overview of Regulatory Mechanisms
Description of ATCase
Allosteric Properties of ATCase

2. Biological Processes are Carefully Regulated
Allosteric Control:
The activity of some proteins can be controlled by modulating
the levels of small signalling molecules. The binding of these
molecules causes conformational changes in the protein
which affect its activity.
Multiple forms of Enzymes:
Different tissues or developmental stages sometimes have specific
versions of a given enzyme which have distinct properties although
they may have the same basic activity.
Reversible Covalent Modification:
The activity of many proteins is controlled by attachment of small
chemical groups. The most common such modification is
phosphorylation- attachment of a phosphate group.
Proteolytic Activation:
Some enzymes are synthesized in an inactive form and must be
activated by cleavage of the inactive form.

3. Allosteric Regulation
Allosteric enzymes have multiple subunits which exert influence on one
another in the complex. The binding of substrate at one site affects the
affinity for substrates at other sites, eventually causing a conformational
shift from a less activate state to a more active state. (cooperativity)
Allosteric enzymes don’t follow Michaelis-Menten kinetics. The activity
increases steeply above a “threshold” so that a small change in [S]
causes a large change in activity.
Small molecule regulators can bind to the enzyme and change the
threshold, so as to adjust the activity to the required level.
With Inhibitor
With Activator
Less
Active
State
More
Active
State

4. Aspartate Transcarbamoylase
Aspartate transcarbamoylase, or ATCase, catalyses the first step in
a biosynthetic pathway that produces pyrimidine nucleotides (eg CTP)
needed for nucleic acids, energy storage, and enzyme cofactors.
ATCase
Many more enzymes…

5. CTP ATCase is inhibited by the end-product of its pathway ATCase
Aspartate
&
Carbamoyl
phosphate
CTP
(doesn’t resemble
substrates of
ATCase)
This is an example of feedback inhibition. When CTP is abundant,
the pathway is shut down, but when CTP levels are low and more is
needed, the activity of ATCase increases to make more CTP.

6. c r Quaternary Structure of ATCase ATCase has two subunit types:
c subunit (catalytic subunit; 34 kD) which forms trimers
r subunit (regulatory subunit; 17 kD) which forms dimers
These can be dissociated, isolated, and reconstituted.
Active Complex: c6r6 c
Catalytic
Subunit: r
Regulatory
Subunit:

7. Top
View
Side
View

8. Identification of Active Sites using an Inhibitor
The compound PALA is a
structural mimic of an
intermediate in the reaction.
X-ray crystallography
reveals that it binds
at the active site in
between 2 catalytic
subunits.

9. PALA Binding causes a Conformational Change
Two distinct quaternary forms exist in equilibrium.
PALA stabilizes the more active R state.
T state for “tense”
predominates in
absence of substrate.
R state for “relaxed”
predominates in
presence of substrate.

10. CTP Binding inhibits the Conformational Change
CTP binds in regulatory sites on the r subunits, distant from
the active sites.
The allosteric inhibitor CTP shifts the equilibrium toward the
less active T state.

11. The R to T transition is All-or-Nothing
In ATCase, a given complex is either in the R state or T state- there
are no “mixed” complexes.
But there is a mixed population of T-state complexes and R-state
complexes at equilibrium.

12. [T]eq R T = 200 = Keq [R]eq RT ln ( Keq )
Thermodynamics of the Allosteric Transition
In the absence of substrate or regulators, the T state is about 200
times as prevalent as the R state. (at equilibrium)
[T]eq
R T
= 200 = Keq
[R]eq
R state
RT ln ( Keq )
T state
= 13.1 kJ/mol
So only a small energy difference exists between the T and R states.
The binding of effectors could easily modulate this energy difference.

13. ATCase does not follow Michaelis-Menten Kinetics
ATCase shows a sigmoidal rate profile.

14. T R ATCase switches between T and R states
At low substrate concentrations, the enzyme is primarily in the
less active T state. But as [substrate] increases, more of the
complexes switch to the more active R state.
T R
(T state curve is
identical to isolated
catalytic subunits)
High Km
Low Km

15. T R CTP acts as an Allosteric Inhibitor
CTP acts to shift the equilibrium towards the T states, favoring the
High Km form of the enzyme and reducing the overall activity.
CTP
T R
High Km
Low Km

16. T R ATP acts as an Allosteric Activator
CTP acts to shift the equilibrium towards the R states, favoring the
low Km form of the enzyme and increasing the overall activity.
ATP
T R
High Km
Low Km

17. Binding of Effectors Adjusts the Equilibrium between T and R States
[T]eq
[R]eq
R T
In the presence of ATP
a higher percentage
of complexes
are in the T state.
In the presence of CTP
a lower percentage
of the complexes
are in the R state.
= [S] / (Km of R state)

18. The binding of the effector influences the binding of substrate
ATP
Allosteric
Equilibrium
R T
CTP
R + S RS
Substrate-
Binding
Equilibrium
CTP
ATP
T + S TS

19. Physiological Role of CTP and ATP Regulation of ATCase
CTP is a feedback inhibitor. When CTP levels are high, it is
unnecessary to make more pyrimidines, so the inhibition
of ATCase slows down the pathway. When CTP levels fall,
the inhibition is removed, and more pyrimidines
can be synthesized.
ATP is a purine nucleotide and is not a product of the
ATCase pathway. ATP is the major cellular energy source
and if ATP levels are high, the cell is metabolically very
active and preparing to divide. Therefore it must duplicate
its DNA, and both ATP and CTP are needed for DNA synthesis.
So high ATP levels can override the inhibitory affects of CTP.

20. Summary:
Aspartate transcarbamoylase (ATCase) is an allosteric enzyme which
carries out the first step in the synthesis of pyrimidine nucleotides.
Allosteric enzymes use changes in conformation to switch between
different states which have different levels of activity.
Binding of allosteric effectors can control the switch between states,
and thereby increase or decrease the enzyme activity to exert control over biological processes.
Key Concepts:
Types of Regulation
Feedback inhibition
Allosteric transition in ATCase
ATP and CTP as allosteric effectors of ATCase