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 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

ATCase Consists of Separate Catalytic and Regulatory Subunits

• Can be separated into regulatory and catalytic subunits by treatment with p-hydroxy-mercuribenzoate, which reacts with sulfhydryl groups

2c3 + 3r2 c6r6

Ultracentrifugation Activity

PCMBS treated

ACTaseNative ACTase11.6S

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 – PCMBS displaces Zn and destabilizes the domain

Structure of ATCase

Potent competitiveinhibitor

Carbamoyl phosphate

Aspartate

Use of PALA to locate active site

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

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 modulatethe T-to-R equilibrium

CTP is an allosteric inhibitor

T>R

ATP is an allosteric activator

High purine

mRNA synthesis ↑

R>T

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+

Proximal histidine

Sixth Co-ordination site

Oxygen binding changes the position of the iron ion

Fifth Co-ordination site

Myoglobin – stabilising bound oxygen

Why is haemoglobin more efficient at binding oxygen?

11 and 22 dimers

Quaternary structure of deoxyhemoglobin - HbA

Oxygen binding to myoglobin

Simple equilibrium.

Haemoglobin as an allosteric protein

• Haemoglobin consists of 2 and 2 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

• Assumes1. 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 11 wrt 22 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 extremelyunstable

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

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

deoxygenated

Β chains

Β chain mutation

Plasmodium falciparum

Why is HbS so prevalent in Africa

• Sickle cell trait (one allele mutation) resistant to malaria