Regulatory Strategies: ATCase & Haemoglobin

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-mercuribenzoate, 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 R>T T>R

17. 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. Why does ATCase display sigmoidal kinetics

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 T>R

21. ATP is an allosteric activator R>T High purine mRNA synthesis ↑

22. Haemoglobin

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 doesn’t 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 αβ dimers favours release of O2 by favouring the T state

49. Carbon dioxide promotes the release of oxygen

50. Sickle cell anaemia