Transcript Biochemistry 6/e - Personal Webspace for QMUL

Regulatory Strategies: ATCase &
Haemoglobin
Aspartate transcarbamolase is allosterically inhibited by the end
product of its pathway
Carbamoyl phosphate + aspartate  N-carbamoylaspartate + Pi
Aspartate transcarbamolase
• Catalyses the first step (the committed step)
in the biosynthesis of pyrimidines (thiamine
and cytosine), bases that are components of
nucleic acids
Condensation of aspartate and carbomyl phosphate to form NCarbamoylaspartate
• How is the enzyme regulated to generate
precisely the amount of CTP needed by the
cell?
CTP inhibits ATCase, despite
having little structural similarity to
reactants or products
ATCase Consists of Separate Catalytic
and Regulatory Subunits
• Can be separated
into regulatory and
catalytic subunits
by treatment with
p-hydroxymercuribenzoate,
which reacts with
sulfhydryl groups
Mercurial dissociate ATCase into
two subunits
Native ACTase
11.6S
PCMBS treated
2.8S 5.8S
2c3 + 3r2  c6r6
Ultracentrifugation
Activity
ACTase
Subunit characteristics
• Regulatory subunit (r2)
– Two chains (17kd each)
– Binds CTP
– No enzyme activity
• Catalytic subunit (c3)
– Three chains
– Retains enzyme activity
– No response to CTP
Structure of ATCase
Cysteine binds Zn – PCMBS
displaces Zn and destabilizes
the domain
Use of PALA to locate active site
Carbamoyl phosphate
Aspartate
Potent
competitive
inhibitor
Active site of ATCase
The T-to-R state transition
Each catalytic trimer has 3 substrate binding sites
Enzyme has two quaternary forms.
CTP stabilises the T state
• T state when CTP bound
• Binding site for CTP
in each regulatory domain
• Binds 50Å from active site
– allosteric
R and T state are in equilibrium
Mechanism for CTP inhibition
ATCase displays sigmoidal kinetics
Cooperativity
R>T
T>R
Why does ATCase display
sigmoidal kinetics
• The importance of the changes in quaternary
structure in determining the sigmoidal curve is
illustrated by studies on the isolated catalytic trimer,
freed by p-hydroxymercuribenzoate treatment.
• The catalytic subunit shows Michaelis-Menten
kinetics with kinetic parameters indistinguishable
from those deduced for the R-state.
• The term tense is apt – the regulatory dimers hold
the two catalytic trimers close so key loops collide
& interfere with the conformational adjustments
necessary for high affinity binding & catalysis.
Basis for the sigmoidal curve
(mixture of two Michaelis Menten enzymes)
Low KM
High KM
Allosteric regulators modulate
the T-to-R equilibrium
CTP is an allosteric inhibitor
T>R
ATP is an allosteric activator
R>T
High purine
mRNA synthesis ↑
Haemoglobin
Myoglobin
• Myoglobin is a single
polypeptide,
hemoglobin has four
polypeptide chains.
• Haemoglobin is a
much more efficient
oxygen-carrying
protein. Why?
Myoglobin and Haemoglobin
bind oxygen at iron atoms in
heme
1
2
Fe2+
3
4
Oxygen binding changes the
position of the iron ion
Sixth
Co-ordination site
Fifth
Co-ordination site
Proximal histidine
Myoglobin – stabilising bound
oxygen
Why is haemoglobin more
efficient at binding oxygen?
Quaternary structure of
deoxyhemoglobin - HbA
a1b1 and a2b2 dimers
Oxygen binding to myoglobin
Simple equilibrium.
Haemoglobin as an allosteric protein
• Haemoglobin consists of 2a and 2b chains
• Each chain has an oxygen binding site,
therefore haemoglobin can bind 4 molecules
of oxygen in total
• The oxygen-binding characteristics of
haemoglobin show it to be allosteric
Oxygen binding to haemoglobin
in rbc
Cooperativity
Cooperative unloading of oxygen
enhances oxygen delivery
Haemoglobin
• Two principal models have been developed
to explain how allosteric interactions give
rise to sigmoidal binding curves
• The concerted model
• The sequential model
Concerted model
• Oxygen can bind to either conformation, but
as the number of sites with oxygen bound
increases, so the equilibrium becomes
biased towards one conformation (in the
case of increasing oxygen bound, the R
conformation)
Concerted model
• Developed by Jacques Monod, Jeffries Wyman and
Jeanne-Pierre Changeaux in 1965
• In this model all the polypeptide chains must be in an
equilibrium that enables two possible conformations to
exist
Concerted model
• The concerted model assumes:
1. The protein interconverts between the two
conformation T and R but all subunits must be in
the same conformation
2. Ligands bind with low affinity to the T state and
high affinity to the R state
3. Binding of each ligand increases the probability
that all subunits in that protein molecule will be
in the R state
Sequential model
• Assumes
1. Each polypeptide chain can only adopt one of
two conformations T and R.
2. Binding of ligand switches the conformation of
only the subunit bound.
3. Conformational change in this subunit alters the
binding affinity of a neighbouring subunit i.e. a
T subunit in a TR pair has higher affinity that in
a TT pair because the TR subunit interface is
different from the TT subunit interface.
Sequential model
• Devised by Dan Koshland in the 1950s
• Substrate binds to one site and causes the polypeptide to
change conformation
• Substrate binding to the first site affects the binding of a
second substrate to an adjoining site
• And so on for other binding sites …
How does oxygen binding induce
change from T to R state
Quaternary structural changes on oxygen
binding (T  R)
Rotation of a1b1 wrt a2b2 dimers
Conformational change in haemoglobin
T→ R
The role of 2,3
bisphosphoglycerate in red blood
cells
Haemoglobin must remain in T
state in absence of oxygen
T – state is extremely
unstable
2,3-BPG (an allosteric effector) binds &
stabilizes the T state (released in R state)
Fetal haemoglobin doesn’t bind 2,3-BPG
so well so has higher oxygen affinity
Bohr effect (protons are also allosteric
effectors)
T-state stabilized by
salt bridges
Salt bridges
Thus oxygen is
released
Carbonic anhydrase
Also … CO2 forms carbamate (R-NH-CO2) with N-ter – at
interface between αβ dimers favours release of O2 by
favouring the T state
Carbon dioxide promotes the release of
oxygen
Sickle cell anaemia
Β chain mutation
Β chains
deoxygenated
Why is HbS so prevalent in
Africa
Plasmodium falciparum
• Sickle cell trait (one allele mutation)
resistant to malaria