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. Native ACTase PCMBS treated ACTase 2c3 + 3r2 c6r6 Ultracentrifugation

9. Cysteine binds Zn – PCMBS displaces Zn and destabilizes the domain

10. Carbamoyl phosphate Aspartate Potent competitive inhibitor

15. ATCase displays sigmoidal kinetics

16. 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-Menton 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.

17. Basis for the sigmoidal curve

18. CTP is an allosteric inhibitor

19. ATP is an allosteric activator

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

25. a1b1 and a2b2 dimers

26. Oxygen binding to myoglobin

27. 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

28. Oxygen binding to haemoglobin

29. Cooperativity enhances oxygen delivery

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

31. 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)

32. 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

33. 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

34. 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.

35. 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 …

36. Quaternary structural changes (R  T) Rotation of a1b1 wrt a2b2 dimers

37. Conformational change in haemoglobin

39. 2,3-BPG (an allosteric effector) binds & stabilizes the T state (released in R state)

40. Fetal haemoglobin doesn’t bind 2,3-BPG so well so has higher oxygen affinity

41. Bohr effect (protons are also allosteric effectors) T-state stabilized

42. Carbonic anhydrase Also … CO2 forms carbamate (R-NH-CO2) with N-ter – favours release of O2 by favouring the T state

43. Carbon dioxide promotes the release of oxygen

45. deoxygenated

46. Plasmodium falciparum