Regulatory Strategies: ATCase

1
Regulatory Strategies ATCase Haemoglobin
2
Aspartate transcarbamolase is allosterically
inhibited by the end product of its pathway
Carbamoyl phosphate aspartate ?
N-carbamoylaspartate Pi
3
Aspartate transcarbamolase

• Catalyses the first step (the committed step) in
  the biosynthesis of pyrimidines (thiamine and
  cytosine), bases that are components of nucleic
  acids

4
Condensation of aspartate and carbomyl phosphate
to form N-Carbamoylaspartate
5

• How is the enzyme regulated to generate precisely
  the amount of CTP needed by the cell?

6
CTP inhibits ATCase, despite having little
structural similarity to reactants or products
7
ATCase Consists of Separate Catalytic and
Regulatory Subunits

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

8
Mercurial dissociate ATCase into two subunits
11.6S
PCMBS treated ACTase
Native ACTase
2.8S 5.8S
2c3 3r2 ? c6r6
Ultracentrifugation Activity
9
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

10
Structure of ATCase
Cysteine binds Zn PCMBS displaces Zn and
destabilizes the domain
11
Use of PALA to locate active site
Carbamoyl phosphate
Aspartate
Potent competitive inhibitor
12
Active site of ATCase
13
The T-to-R state transition
Each catalytic trimer has 3 substrate binding
sites Enzyme has two quaternary forms.
14
CTP stabilises the T state

• T state when CTP bound
• Binding site for CTP
• in each regulatory domain
• Binds 50Å from active site
• allosteric

15
R and T state are in equilibrium
Mechanism for CTP inhibition
16
ATCase displays sigmoidal kinetics
Cooperativity
RgtT
TgtR
17
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.

18
Basis for the sigmoidal curve(mixture of two
Michaelis Menten enzymes)
Low KM
High KM
19
Allosteric regulators modulatethe T-to-R
equilibrium
20
CTP is an allosteric inhibitor
TgtR
21
ATP is an allosteric activator
RgtT
High purine mRNA synthesis ?
22
Haemoglobin
23
(No Transcript)
24
Myoglobin

• Myoglobin is a single polypeptide, hemoglobin has
  four polypeptide chains.
• Haemoglobin is a much more efficient
  oxygen-carrying protein. Why?

25
Myoglobin and Haemoglobin bind oxygen at iron
atoms in heme
1
2
Fe2
3
4
26
Oxygen binding changes the position of the iron
ion
Sixth Co-ordination site
Fifth Co-ordination site
Proximal histidine
27
Myoglobin stabilising bound oxygen
28
Why is haemoglobin more efficient at binding
oxygen?
29
Quaternary structure of deoxyhemoglobin - HbA
a1b1 and a2b2 dimers
30
Oxygen binding to myoglobin
Simple equilibrium.
31
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

32
Oxygen binding to haemoglobin in rbc
Cooperativity
33
Cooperative unloading of oxygen enhances oxygen
delivery
34
Haemoglobin

• Two principal models have been developed to
  explain how allosteric interactions give rise to
  sigmoidal binding curves
• The concerted model
• The sequential model

35
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)

36
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

37
Concerted model

• The concerted model assumes
• The protein interconverts between the two
  conformation T and R but all subunits must be in
  the same conformation
• Ligands bind with low affinity to the T state and
  high affinity to the R state
• Binding of each ligand increases the probability
  that all subunits in that protein molecule will
  be in the R state

38
Sequential model

• Assumes
• Each polypeptide chain can only adopt one of two
  conformations T and R.
• Binding of ligand switches the conformation of
  only the subunit bound.
• 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.

39
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

40
How does oxygen binding induce change from T to R
state
41
Quaternary structural changes on oxygen binding
(T ? R)
Rotation of a1b1 wrt a2b2 dimers
42
Conformational change in haemoglobin
T ? R
43
The role of 2,3 bisphosphoglycerate in red blood
cells
44
Haemoglobin must remain in T state in absence of
oxygen
T state is extremely unstable
45
2,3-BPG (an allosteric effector) binds
stabilizes the T state (released in R state)
46
Fetal haemoglobin doesnt bind 2,3-BPG so well so
has higher oxygen affinity
47
Bohr effect (protons are also allosteric
effectors)
T-state stabilized by salt bridges
Salt bridges
Thus oxygen is released
48
Carbonic anhydrase
Also CO2 forms carbamate (R-NH-CO2) with N-ter
at interface between aß dimers favours release
of O2 by favouring the T state
49
Carbon dioxide promotes the release of oxygen
50
Sickle cell anaemia
51
? chain mutation
? chains
deoxygenated
52
Why is HbS so prevalent in Africa

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

Plasmodium falciparum