Lecture 14:Regulation of Proteins 1:

Allosteric Control of ATCase

Overview of Regulatory Mechanisms

Description of ATCase

Allosteric Properties of ATCase

Biological Processes are Carefully Regulated

Allosteric Control: The activity of some proteins can be controlled by modulatingthe levels of small signalling molecules. The binding of thesemolecules causes conformational changes in the proteinwhich affect its activity.

Multiple forms of Enzymes:Different tissues or developmental stages sometimes have specificversions of a given enzyme which have distinct properties althoughthey may have the same basic activity.

Reversible Covalent Modification:The activity of many proteins is controlled by attachment of smallchemical groups. The most common such modification isphosphorylation- attachment of a phosphate group.

Proteolytic Activation:Some enzymes are synthesized in an inactive form and must beactivated by cleavage of the inactive form.

Allosteric Regulation

LessActiveState

MoreActiveState

With Inhibitor

With Activator

Allosteric enzymes have multiple subunits which exert influence on oneanother in the complex. The binding of substrate at one site affects theaffinity for substrates at other sites, eventually causing a conformationalshift from a less activate state to a more active state. (cooperativity)

Allosteric enzymes don’t follow Michaelis-Menten kinetics. The activityincreases 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 thethreshold, so as to adjust the activity to the required level.

Aspartate Transcarbamoylase

Aspartate transcarbamoylase, or ATCase, catalyses the first step ina biosynthetic pathway that produces pyrimidine nucleotides (eg CTP)needed for nucleic acids, energy storage, and enzyme cofactors.

ATCase Many more enzymes…

ATCase is inhibited by the end-product of its pathway

ATCaseAspartate

&Carbamoylphosphate

CTP

(doesn’t resemblesubstrates ofATCase)

This is an example of feedback inhibition. When CTP is abundant,the pathway is shut down, but when CTP levels are low and more isneeded, the activity of ATCase increases to make more CTP.

Quaternary Structure of ATCase

ATCase has two subunit types:c subunit (catalytic subunit; 34 kD) which forms trimersr subunit (regulatory subunit; 17 kD) which forms dimers

These can be dissociated, isolated, and reconstituted.

c c

c

r

r

CatalyticSubunit:

RegulatorySubunit:

Active Complex: c6r6

TopView

SideView

Identification of Active Sites using an Inhibitor

The compound PALA is a structural mimic of an intermediate in the reaction.

X-ray crystallographyreveals that it bindsat the active site inbetween 2 catalyticsubunits.

PALA Binding causes a Conformational Change

T state for “tense”predominates inabsence of substrate.

R state for “relaxed”predominates inpresence of substrate.

Two distinct quaternary forms exist in equilibrium.PALA stabilizes the more active R state.

CTP Binding inhibits the Conformational Change

CTP binds in regulatory sites on the r subunits, distant fromthe active sites.

The allosteric inhibitor CTP shifts the equilibrium toward theless active T state.

The R to T transition is All-or-Nothing

In ATCase, a given complex is either in the R state or T state- thereare no “mixed” complexes.

But there is a mixed population of T-state complexes and R-statecomplexes at equilibrium.

Thermodynamics of the Allosteric Transition

In the absence of substrate or regulators, the T state is about 200times as prevalent as the R state. (at equilibrium)

R T[T]eq

[R]eq

= 200 = Keq

T state

R state

RT ln ( Keq )

= 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.

ATCase does not follow Michaelis-Menten Kinetics

ATCase shows a sigmoidal rate profile.

ATCase switches between T and R states

At low substrate concentrations, the enzyme is primarily in theless active T state. But as [substrate] increases, more of thecomplexes switch to the more active R state.

High Km Low Km

T R

(T state curve isidentical to isolatedcatalytic subunits)

CTP acts as an Allosteric Inhibitor

High Km Low Km

T RCTP

CTP acts to shift the equilibrium towards the T states, favoring theHigh Km form of the enzyme and reducing the overall activity.

ATP acts as an Allosteric Activator

High Km Low Km

T RATP

CTP acts to shift the equilibrium towards the R states, favoring thelow Km form of the enzyme and increasing the overall activity.

Binding of Effectors Adjusts the Equilibrium between T and R States

= [S] / (Km of R state)

L = [T]eq

[R]eq

In the presence of ATPa higher percentageof complexesare in the T state.

In the presence of CTPa lower percentageof the complexesare in the R state.

R T

The binding of the effector influences the binding of substrate

R T

T + S TS

R + S RS

ATP

CTP

ATPCTP

AllostericEquilibrium

Substrate-Binding

Equilibrium

Physiological Role of CTP and ATP Regulation of ATCase

CTP is a feedback inhibitor. When CTP levels are high, it isunnecessary to make more pyrimidines, so the inhibitionof ATCase slows down the pathway. When CTP levels fall,the inhibition is removed, and more pyrimidinescan be synthesized.

ATP is a purine nucleotide and is not a product of theATCase pathway. ATP is the major cellular energy sourceand if ATP levels are high, the cell is metabolically veryactive and preparing to divide. Therefore it must duplicateits DNA, and both ATP and CTP are needed for DNA synthesis.

So high ATP levels can override the inhibitory affects of CTP.

Summary:

Aspartate transcarbamoylase (ATCase) is an allosteric enzyme whichcarries out the first step in the synthesis of pyrimidine nucleotides.

Allosteric enzymes use changes in conformation to switch betweendifferent 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 exertcontrol over biological processes.

Key Concepts:

Types of RegulationFeedback inhibitionAllosteric transition in ATCaseATP and CTP as allosteric effectors of ATCase