Regulatory Strategies: ATCase &

Haemoglobin

Allosteric Regulation

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 N-

Carbamoylaspartate

• 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

CTP inhibits ATCase, despite

having little structural similarity to

reactants or products

FEEDBACK INHIBITION

Allosteric regulation

ATCase Structure

ATCase Consists of Separate Catalytic

and Regulatory Subunits

• Can be separated

into regulatory and

catalytic subunits

by treatment with

p-hydroxy-

mercuribenzoate

(PCMB), which

reacts with

sulfhydryl groups

2c3 + 3r2 c6r6

Ultracentrifugation Activity

PCMB treated

ACTase Native ACTase 11.6S

2.8S 5.8S

Mercurial dissociate ATCase into

two subunits

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

Cysteine binds Zn – PCMB

displaces Zn and destabilizes

the domain

Structure of ATCase

Potent

competitive

inhibitor

Carbamoyl phosphate

Aspartate

Use of PALA to locate active site

Active site of ATCase

PALA

C1

C2

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

T>R

R>T

Cooperativity

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)

High KM

Low KM

Allosteric regulators modulate

the T-to-R equilibrium

CTP is an allosteric inhibitor

T>R

ATP is an allosteric activator

High purine

High Energy

mRNA synthesis ↑

R>T

ATP Competes for same binding site

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

3 4

Fe2+

Fe has 6 co-ordination sites

Proximal histidine

Sixth

Co-ordination site

Oxygen binding changes the

position of the iron ion

Fifth

Co-ordination site Electron

rearrangement

Myoglobin – stabilising bound

oxygen

Stronger binding

Why is haemoglobin more

efficient at binding oxygen?

a1b1 and a2b2 dimers

Quaternary structure of

deoxyhemoglobin - HbA

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

Physiological significance of

allosteric regulation

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

N

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)

Requires increased oxygen for T to R transition

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

Thus oxygen is

released

Salt bridges

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

Aggregates when in deoxygenated form

Β chains

Β chain mutation

Plasmodium falciparum

Why is HbS so prevalent in

Africa

• Sickle cell trait (one allele mutation)

resistant to malaria