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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
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 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