Lecture 13 Allostery

Question 1

What are the mechanisms of enzyme regulation?

Answer 1

• Allostery
• Multiple Isozymes (same reaction different protein)
• Covalent modification
• Proteolytic activation
• Altering amount of enzyme (regulated synthesis and degradation)

Question 2

How do allosteric enzymes work?

Answer 2

• Allosteric enzyme have distinct regulatory sites and multiple active sites
• The binding of small molecules to regulatory sites controls enzyme activity
• These small regulatory molecules are often products of the pathway in which the enzyme functions
• Allosteric enzymes often show cooperativity, binding to one site affects others

Question 3

What is Aspartate Transcarbamoylase?

Answer 3

• Sometimes called Aspartate Carbamoyl Transferease
• First committed step in synthesis of pyrimidine nucleotides (CTP, UTP and TTP)
• Catalyses the condensation of Carbomoyl phosphate and Aspartate to give N-Carbomoylaspartate

Question 4

How is Aspartate Transcarbamoylase made?

Answer 4

Carbamoyl phosphate + aspartate = N-carbamoylaspartate

Question 5

What kinetics does Aspartate Transcarbamoylase show?

Answer 5

• ATCase does not show Michelis-Menten kinetics

- Shows sigmoidal kinetics

Question 6

How is Pyrimidine (and Purine) Biosynthesis regulated?

Answer 6

• Biosynthesis of pyrimidines (and purines) essential for the production of DNA
• Pathway must be very tightly regulated otherwise DNA synthesis will be impaired
• Multiple enzymes in both pathways are tightly regulated

Question 7

How is Aspartate Transcarbamoylase regulated?

Answer 7

• Aspartate Transcarbamoylase Allosterically regulated by end product of purine biosynthetic pathway, CTP
• Low CTP high ATCase activity
• High CTP, low ATCase activity
• Feedback Inhibition
• as CTP increases rate of N-carbamoylaspartate formation decreases

Question 8

What does CTP look like?

Answer 8

• CTP looks nothing like carbomoyl phosphate, aspartate or N-carbomoylasparate
• much larger molecule with three phosphate tails.

Question 9

What does ATCase consist of ?

Answer 9

-ATCase consists of multiple subunits
-Demonstrated using using ultracentrifugation after treatment with of p-hydroxymercuribenzoate
-c subunit (3 polypeptides) has catalytic activity but is not responsive to CTP
-r subunit (2 polypeptides) binds CTP but has no catalytic activity
-Native conformation is:
2C3 +3r2 = c6r6

Question 10

How does CTP inhibit ATCase?

Answer 10

-Active site identified by using PALA a transition state analogue that strongly inhibits ATCase
-PALA binds to active sites site located between pairs of c chains
-Binding of PALA causes c subunits to move 12 Å apart and to rotate 10° along axis of symmetry
-r subunits rotate by 15° to accommodate change in c subunits
-Two forms of enzyme called T (tense) and R (relaxed)
-Substrate binds more efficiently to R state
-Shows cooperative binding in that substrate bound to one active site increases affinity of binding to other active sites
-CTP inhibits by stabilizing the T state making the enzyme less active
(T state-less active R state-more active)

Question 11

What is CTP to ACTase?

Answer 11

CTP is allosteric inhibitor

Question 12

What is ATP to ACTase?

Answer 12

ATP is allosteric activator

Question 13

What is haemoglobin?

Answer 13

• Hemoglobin in North American textbooks
• Main oxygen-carrying protein in human body
• Found in red blood cells
• Binds Oxygen in lungs and releases it in tissues
• Also binds CO2 in tissues for release in lungs

Question 14

What is the structure of heamoglobin?

Answer 14

• Haemoglobin is tetrameric protein with 2 identical alpha subunits and 2 identical beta subunits
• Structure has a α2β2 configuration
• Each globin subunit contains a Haem (Heme) group which binds O2 (4 O2 in total)
• The Haem cofactor is a Tetrapyrrole ring molecule in association with an Fe2+ ion

Question 15

Is haemoglobin a good oxygen carrier?

Answer 15

• Tetrameric Haemoglobin is a much more efficient oxygen carrier than related monomeric protein myoglobin
• Due to cooperative binding to O2 to Haemoglobin

Question 16

How does oxygen bind to haem?

Answer 16

• The molecular mechanism for O2 binding to Haem was elucidated using Myoglobin
• Mechanism for Myoglobin same as occurs in single Haemoglobin subunit
• Fe2+ lies outside plane of tetrapyrrole ring
• On binding Fe2+ moves into plane of ring
• A distal Histidine residue helps to stabilize the bound O2

Question 17

How does oxygen bind to haemoglobin?

Answer 17

• O2 binding is cooperative
• Binding of O2 Haem in one subunit increases affinity of O2 to others
• Binding of last O2 is approximately 200 times more efficient than binding of first O2
• Cooperativity also applies to O2 unloading

Question 18

How does haemoglobin change when it binds to oxygen?

Answer 18

-O2 causes change in tertiary structure
-α1β1 dimers rotate relative to α2β2 by 15°
-The deoxygenated form is called the Tense (T) state
-The oxygenated forms is called the Relaxed (R) state
-In R state O2 binding sites are open and are more able to bind O2
Deoxyhemoglobin= Oxyhemoglobin

Question 19

What models exist to explain cooperativity in oxygen binding in haemoglobin?

Answer 19

2 models to explain cooperativity in O2 binding:

• Concerted model
• Sequential model

Question 20

How does the concerted model work?

Answer 20

-All O2 bound forms of haemoglobin in equilibrium between T and R states
-As more O2 binds equilibrium shifts towards R state
(shift to R form can happen with any numbe of bonded O)

Question 21

How does the sequential model work?

Answer 21

• Binding of O2 one subunit causes shift to R form
• Allows neighboring subunit to bind O2 with slightly higher affinity (one bonds,shift to R form, then the next one can bond and so on)

Question 22

What is 2,3 Bisphosphoglycerate and what is it used for?

Answer 22

• Pure Haemoglobin binds O2 much more strongly than Haemoglobin in blood
• Important that T-state must be stabilized or Haemoglobin would never release O2
• In erythrocytes there is allosteric regulator called 2,3 Bisphosphoglycerate (2,3 BPG)
• 2,3 BPG binds to T form and stabilizes it

Question 23

What difference is there in fetal haemoglobin and maternal haemoglobin?

Answer 23

Oxygen flows from maternal oxyhaemoglobin to fetal deoxyhaemoglobin, fetal haemoglobin binds more readily.

Question 24

What role do carbon dioxide and hydrogen ions play in oxygen binding?

Answer 24

-CO2 and H+ are allosteric regulators of O2 binding
-Increased CO2 and decreased pH both reduce affinity for O2 by stabilizing T state
-“Bohr” effect
(higher pH,so more hydrogen ions, Oxygen binds more readily, however higher carbon dioxide concentration oxygen binds less to haemoglobin)

Question 25

What effect does decreased pH have on the state of haemoglobin?

Answer 25

-Decreased pH stabilizes T state by allowing formation of salt bridges within globin subunits

Question 26

What effect does increase CO2 concentration have on haemoglobin?

Answer 26

• Increased CO2 leads to decreased pH through action of carbonic anhydrase
• CO2 also directly effects quaternary structure by reacting with amino termini to form negatively charged carbamate
• Amino termini of subunits lie at αβ interface
• Carbmate groups allow formation of salt bridges that stabilize T state

Question 27

Summary of allosteric regulation.

Answer 27

• Allosteric enzymes do not show Michealis-Menten kinetics and often show cooperative regulation
• Allosteric enzyme consist of several subunits with multiple active and regulatory sites
• Aspartate Transcarbamylase is allosterically regulated by CTP and ATP
• Binding of oxygen to haemoglobin is cooperative and allosterically regulated by 2,3 BPG, CO2 and pH